Sophie Monnot

Nadine Gigarel

David C Samuels

Philippe Burlet

Laetitia Hesters

Nelly Frydman

René Frydman

Violaine Kerbrat

Benoît Funalot

Jéléna Martinovic

Hesters

Benoit Funalot

Martinovic

Alexandra Benachi

Josué Feingold

Arnold Munnich

Bonnefont

Julie Steffann

Segregation of mtDNA Throughout Human Embryofetal Development: m.3243A>G as a Model System

Keywords: mitochondrial DNA, MELAS, m.3243A>G, embryo, mitochondria Mitochondria, mitochondrial DNA, MELAS, NARP, respiratory chain deficiency, 23 embryo, preimplantation genetic diagnosis 24 25

come

Introduction 1 2
Mitochondrial DNA (mtDNA, Genbank NC_001807.4) disorders are common 3 diseases with maternal inheritance. Their high clinical variability is explained, at least 4 in part, by variation of the mutant load (heteroplasmy) among individuals, and also 5 across organs and tissues within an individual, so that mutant loads will surpass the 6 tissue specific threshold, leading to the manifestation of symptom (Chinnery et al., 7 1997; [START_REF] Jeppesen | 11 Muscle phenotype and mutation load in 51 persons with the 3243A>G 12 mitochondrial DNA mutation[END_REF]. The usual severity of these diseases, the recurrence 8 risk, usually high though being difficult to predict at the individual level, in offspring 9 from heteroplasmic carriers [START_REF] Chinnery | Genetic counseling and 3 prenatal diagnosis for mtDNA disease[END_REF], and the absence of efficient 10 therapy, commonly result in a request from at-risk couples for prenatal (PND) or 11 preimplantation diagnosis (PGD). Such procedures are however hampered by the 12 lack of data regarding mtDNA segregation during embryofetal development . 13 In this respect, most available data come from animal studies. Dramatic shifts 14 of mtDNA populations (variant/wild-type mtDNA ratio) between the mother and her 15 offspring in cattle have suggested that a tight bottleneck might account for such a 16 rapid segregation [START_REF] Laipis | Unequal partitioning of bovine 14 mitochondrial genotypes among siblings[END_REF]. One important function of this bottleneck could 17 be to ensure that new mutations will either be lost or fixed rapidly within individuals, 18 and thus exposed to selection at the population level [START_REF] Bergstrom | Germline bottlenecks and the evolutionary 3 maintenance of mitochondrial genomes[END_REF]19 Roze et al., 2005). Studies in mice have identified the bottleneck during oogenesis 20 [START_REF] Jenuth | Random genetic drift in the 8 female germline explains the rapid segregation of mammalian mitochondrial 9 DNA[END_REF], through either a reduction of mtDNA content in primordial germ 21 cells (PGC, [START_REF] Cree | A reduction of mitochondrial DNA molecules 17 during embryogenesis explains the rapid segregation of genotypes[END_REF], or selection of a small effective number of segregation 22 units for mtDNA [START_REF] Cao | The mitochondrial bottleneck occurs without reduction of mtDNA 17 content in female mouse germ cells[END_REF][START_REF] Cao | New evidence 19 confirms that the mitochondrial bottleneck is generated without reduction of 20 mitochondrial DNA content in early primordial germ cells of mice[END_REF][START_REF] Wai | The mitochondrial DNA genetic bottleneck 9 results from replication of a subpopulation of genomes[END_REF]. There are 23 however no available data so far regarding mutant mtDNA segregation in somatic 24 tissues during embryofetal development in animal models. In humans, a small number of studies of primary oocytes carrying mtDNA 1 deletions [START_REF] Marchington | Evidence 6 from human oocytes for a genetic bottleneck in an mtDNA disease[END_REF] or point mutations [START_REF] Blok | Skewed segregation of the mtDNA 5 nt 8993 (T-->G) mutation in human oocytes[END_REF]Brown et 2 al., 2001;[START_REF] Marchington | Information for genetic management 14 of mtDNA disease: sampling pathogenic mtDNA mutants in the human 15 germline and in placenta[END_REF] have suggested that a bottleneck operates during 3 oogenesis. Random genetic drift appeared as the principal mechanism determining 4 the level of mutant mtDNA within primary oocytes from a carrier of the most common 5 mtDNA mutation, m.3243A>G [START_REF] Brown | Random 13 genetic drift determines the level of mutant mtDNA in human primary oocytes[END_REF]. This mutation affects the MT- TL1 6 gene [mitochondrially encoded tRNA leucine 1 (UUA/G), MIM 590050], and is 7 responsible for the MELAS (Mitochondrial myopathy, encephalopathy, lactic acidosis, 8 and stroke-like episodes, MIM 540000, [START_REF] Goto | 23 Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like 24 episodes (MELAS): a correlative study of the clinical features and 25 mitochondrial DNA mutation[END_REF] and diabetes-deafness 9 syndromes (MIM 520000, van den [START_REF] Van Den Ouweland | Mutation in 6 mitochondrial tRNA(Leu)(UUR) gene in a large pedigree with maternally 7 transmitted type II diabetes mellitus and deafness[END_REF][START_REF] Manouvrier | Point 2 mutation of the mitochondrial tRNA(Leu) gene (A 3243 G) in maternally 3 inherited hypertrophic cardiomyopathy, diabetes mellitus, renal failure, and 4 sensorineural deafness[END_REF]. 10

It is so far not known whether m.3243A>G segregation is governed by random 11 drift in somatic tissues during human embryofetogenesis. The few available studies 12 on m.3243A>G carrier fetuses reported a uniform distribution of mutation load among 13 different tissues from two heteroplasmic 24 and 25 week-old human fetuses 14 [START_REF] Matthews | Comparison of the relative levels of the 3243 (A-->G) mtDNA mutation 18 in heteroplasmic adult and fetal tissues[END_REF][START_REF] Cardaioli | Heteroplasmy of the 23 A3243G transition of mitochondrial tRNA(Leu(UUR)) in a MELAS case and in 24 a 25-week-old miscarried fetus[END_REF]. Our group found similar mutant loads 15 in chorionic villi and amniocytes from 3 heteroplasmic fetuses [START_REF] Bouchet | Prenatal diagnosis of myopathy, encephalopathy, lactic acidosis, 9 and stroke-like syndrome: contribution to understanding mitochondrial DNA 10 segregation during human embryofetal development[END_REF]. 16 All these data suggest that MELAS mutant molecules do not segregate much 17 between 10 to 25 weeks of gestation. 18

However, we have so far lacked data on the transmission of m.3243A>G by a 19 carrier to early embryos, and subsequently no information on segregation of this 20 mutation throughout the entire period of human embryofetal development has 21 existed. Thanks to our PGD and PND programs, we collected various tissues and 22 cells at various stages of development in embryos and fetuses from m.3243A>G 23 carriers, and investigated local and temporal variation of the mutant load, using a 24 Aggregation of our data in early embryos and fetuses, with those from a 3 primary oocyte study in a m.3243A>G carrier [START_REF] Brown | Random 13 genetic drift determines the level of mutant mtDNA in human primary oocytes[END_REF] indicates that 4 mtDNA segregation is governed by random genetic drift, in germ-and somatic cell 5 lines, throughout the entire period of human intra-uterine development. The size of 6 the bottleneck operating during oogenesis for m.3243A>G is additionally suggested 7 to vary between individuals. Finally, comparison of the current data with those we 8 previously reported for another common mtDNA mutation, m.8993T>G (MT-ATP6, 9

ATP synthase subunit 6 gene, MIM 516060) responsible for the NARP syndrome 10 (Neuropathy, Ataxia, Retinitis Pigmentosa, MIM 551500; [START_REF] Steffann | Analysis 1 of mtDNA variant segregation during early human embryonic development: a 2 tool for successful NARP preimplantation diagnosis[END_REF]11 Steffann et al., 2007) indicates that these two mutations differentially influence 12 mtDNA segregation during oogenesis, while their putative impact on mtDNA 13 segregation appears quite similar in somatic cells during embryofetal development. [START_REF] Bouchet | Prenatal diagnosis of myopathy, encephalopathy, lactic acidosis, 9 and stroke-like syndrome: contribution to understanding mitochondrial DNA 10 segregation during human embryofetal development[END_REF]. 4

Accordingly, we postulated that mutant loads over 60% during prenatal life might be 5 predictive of a serious disorder, while a mutant load below 30% might hopefully be 6 associated with absence or low severity of the disease. The most complex situation 7 was a fetal heteroplasmy value in the intermediate range (30 to 60%), a common 8 finding for the MELAS m.3243A>G mutation in the post-natal period. There is indeed 9 no available data correlating intermediate mutant load in the prenatal period with the 10 postnatal outcome. 11

Seven of the 8 couples (couples 2 to 8) elected to resort to a conventional 12 PND approach at first. Couple 1 declined the option of pregnancy termination (TOP), 13 and thus directly went to PGD, that was also offered to couple 2, who had previously 14 experienced 2 TOP after PND. Both couples were informed that, at this early 15 embryonic stage (Day 3), a number of issues remained unsolved, precluding any 16 conclusive prediction on the pre/postnatal outcome of a carrier embryo. These 17 couples were offered a PND at 16 gestation weeks (GW) to confirm the PGD result in 18 case of pregnancy. 19

20

Materials 21

Postnatal period 22

Mutant loads from carrier females were assessed from blood, and when available, 23 urine and oral mucosa samples. Single lymphocytes were isolated from patients 1, 2, 24 3, 5, and 14 as already described [START_REF] Gigarel | Single cell quantification of the 8993T>G 20 NARP mitochondrial DNA mutation by fluorescent PCR[END_REF].

Early embryos 1

Patients 1 and 2 were subjected to one and three separate PGD cycles, respectively. 2 A standard in vitro fertilization protocol was performed and oocytes were collected 3 and fertilized by intracytoplasmic sperm injection (ICSI). 4

A total of 38 embryos were analyzed. Twenty-three of these embryos were of 5 appropriate quality, and were therefore submitted to a 2-blastomere biopsy at post-6 ICSI day 3 for mutant load assessment, as previously described (Gigarel et al., 7 2004). Briefly, under control by a binocular microscope, the blastomeres were rinsed 8 twice in a drop of PBS supplemented with 0.1%polyvinyl alcohol (Sigma Aldrich, 9

France), using a mouth-controlled, finely pulled glass pipette before being transferred 10 into a transparent microcentrifuge tube containing 3 µl of lysis buffer (Cui et al., 11 1989). A small volume of biopsy medium was used as a PCR negative control for 12 each embryo in order to detect any contamination by exogenous DNA. In the 15 13 remaining embryos, assessment of the mutant load was performed on the embryo in 14 toto. All blastomeres were separately analyzed, when possible (couple 1: embryos 1 15 to 3, and couple 2: embryo 13). Embryo 10 from couple 2 was cultured up to the 16 blastocyst stage (Day 5), enabling a trophectoderm biopsy as described elsewhere 17 [START_REF] Mcarthur | Pregnancies and 20 live births after trophectoderm biopsy and preimplantation genetic testing of 21 human blastocysts[END_REF]. The trophectoderm and inner cell mass were analyzed 18 separately. 19 20

Late embryos and fetuses 21

Prenatal diagnosis was performed in 12 fetuses from 7 carrier women. Fetus 2c 22 resulted from embryo 7 and 11 transfer after PGD (patient 2), while the remaining 23 ones were conceived naturally. Chorionic villi (CV, n=8), amniotic fluids 1 (AF1, n=6), 24 and 2 (AF2, n=1), were sampled at 10, 14, and 30 weeks of gestation (GW), 25 respectively. Individual fetal cells were isolated from CV and AF samples from 6 1 fetuses, as already described [START_REF] Steffann | Stability 5 of the m.8993T->G mtDNA mutation load during human embryofetal 6 development has implications for the feasibility of prenatal diagnosis in NARP 7 syndrome[END_REF], and analyzed separately, to 2 investigate the intercellular variation of heteroplasmy. Cord blood was collected from 3 3 fetuses at birth (fetuses 2c, 3, and 4). Postmortem analyses of various tissues were 4 carried out in two 12 week-old fetuses (fetuses 2a and 2b) and one 19 week-old fetus 5 (5b) after TOP, and in a 20 week-old miscarriage product (fetus 7a). Parental 6 informed consent was obtained for these postmortem analyses. Fetal tissues were 7 carefully dissected, and small tissue biopsies were analyzed separately. Multiple 8 samples were taken from two term placentas (fetuses 2c and 3), and two 12GW 9 placentas (fetuses 2a and 2b). 10 11

Methods

12

DNA extraction 13

DNA was extracted from blood and tissues, using the Nucleon Bacc3 kit (Amersham 14 Biosciences, UK), and a classical phenol extraction method, respectively. 15 16

Quantitative analysis of the m.3243A>G mutant load 17

MELAS mutant load was quantified using a semi-quantitative fluorescent PCR-HaeIII 18 restriction test. 19 Since PCR products analysis using a restriction enzyme is thought to allow the 20 possibility of errors in mutant load assessments, secondary to heteroduplex 21 formation during the PCR process [START_REF] Tanno | Quantitation of 17 mitochondrial DNA carrying tRNALys mutation in MERRF patients[END_REF], we first validated our method 22 of heteroplasmy assessment using mixing experiments and standard curve analysis. 23

Wild-type and mutant plasmids were mixed to generate 10 samples, each of them 24 comprising 10 6 mtDNA copies, with the m.3243G>A (mutant) target at concentrations 25 ranging from 1 to 100%, which were also used to determine the lowest rate of 1 mutation detection. Each sample was amplified in triplicate, using both "single cell" 2 and "tissue" methods, and PCR products were submitted to HaeIII digestion. 3

We furthermore ascertained the reliability of our test over a wide range of 4 mtDNA copies, from 10 2 to 10 6 , encompassing the mtDNA copy number usually 5 present in isolated cells such as lymphocytes, blastomeres and oocytes. 6

Single cells and embryos were transferred to 3µl alkaline buffer (Cui et al., 7 1989) and lysed by 10 min heating at 65°C. PCR amplification was carried out using 8 the forward primer (5'-TGAGTTCAGACCGGAGTAATC-3') and the reverse 9 fluorescent primer (*) (5'-(6-Fam) CTTAACAACATACCCATGGC-3'). Separate 10 experimental conditions were devised for whole tissue sample and single cell/early 11 embryo analyses. 12 Tissue sample DNA (1ng) was amplified in a 30µl reaction volume containing 13 1.5U Expand Taq DNA Polymerase, 10X PCR buffer 2 (3µl, Roche Diagnostics, 14 Mannheim, Germany), 0.5µM of each primer (Proligo, Paris, France), and 2mM 15 dNTP mix (Roche Diagnostics, Germany). Initial denaturation was carried out at 97°C 16 for 7 min 20s followed by 20 cycles (97°C for 20s, 60°C for 30s, 68°C for 1min 15s) 17 and final extension of 7 min at 68°C. 18

For a single cell or embryo analysis, the PCR reaction contained 3 µl of lysis 19 buffer (lysed cell or negative control), each primer at 0.5µM, master mix 2X (12.5µl, 20 QIAGEN Multiplex PCR kit, QIAGEN S.A, Courtaboeuf, France), and double-distilled 21 water up to a 25-µl final volume. PCR programs were 15 min of denaturation at 95°C, 22 followed by 25, 26, 27, and 30 PCR cycles for whole embryos, trophoblastic cells, 23 blastomeres, and amniocytes, respectively, consisting of 30s at 94°C, 90s at 60°C 24 and 60s at 72°C, with a 30-min final extension at 60°C. The resulting 251-bp fluorescent PCR products (1µl) were digested for 3 hours 1 using 10 units of HaeIII and subsequently submitted to electrophoresis using an 2 automated genetic analyser ABI3130 (Applied Biosystems). Digestion generated 73-3 bp and 170-bp fluorescent fragments for the mutant and wild-type species, 4 respectively. Results were analysed with the Genescan and Genotyper software 5

(Applied Biosystems). The mutant load was calculated by dividing the mutant peak 6 area (73bp) by the sum of normal (171bp) and mutant (73bp) peak areas. Cross 7 hybridization of oligonucleotide primers to nuclear DNA was ruled out by PCR 8 amplification on mtDNA-less Rho0 cells [START_REF] Parfait | Co-amplification of nuclear 23 pseudogenes and assessment of heteroplasmy of mitochondrial DNA 24 mutations[END_REF]. 9 10

Ascertainment of the fetal origin of the CV/AF cells 11

The fetal origin of single cells collected from CV, AF1, and AF2, was ascertained by 12 simultaneous analysis of parental and embryofetal DNA using nuclear (CA) n 13 microsatellite markers (D6S436, D19S559, D19S559, D16S3395, D6S436, and 14 DXS1073 for fetus 2a, 5a, 5b, 6, 8, and 12b, respectively), enabling to check for 15 biparental contribution to the cell genotype, and the absence of fetal cell 16 contamination by maternal DNA [START_REF] Gigarel | Single cell co-amplification of polymorphic markers for the indirect 15 preimplantation genetic diagnosis of hemophilia A, X-linked 16 adrenoleukodystrophy, X-linked hydrocephalus and incontinentia pigmenti loci 17 on Xq28[END_REF]. 17

Amplification of microsatellites markers in single cells required nested PCR. 18

The first "outer" PCR enabled simultaneous amplification of the MELAS mutation and 19 an appropriate microsatellite marker. Briefly, cells were amplified using "single-cell" 20 PCR conditions described above, as a duplex PCR reaction, using 0.5µM of each 21 primer. 3µl of the first amplification product were subsequently mixed with 22µl of 22 "inner" amplification mix, which contained inner fluorescent microsatellite primers at 23 0.5µM, master mix 2X (12.5µl, QIAGEN Multiplex PCR kit, QIAGEN S.A, 24

Sensitivity, linearity, and reproducibility of mutant load quantification 15

Mixing various proportions of wild type and mutant plasmids enabled us to establish 16 that the measured level of heteroplasmy was a linear function of the expected ratio 17 (Figure 1). The correlation coefficient for the observed vs expected proportion of 18 mutant was 0.99 for both "single cell" and "tissue" conditions. Triplicate experiments 19 yielded to similar results (SD<1.7%), and mutant molecules could be detected in a 20 proportion as low as 2% of the total mtDNA amount, thus validating reproducibility 21 and sensitivity of the assay. These results were got with a 10 6 mtDNA copy number 22

In order to demonstrate an insensitivity to template input amounts for this particular 23 method, we ascertained the reliability of our test over a wide range of mtDNA copies, 24 from 10 2 to 10 6 . Results are displayed in Supp. Figure S1.

Analysis in carrier women 2

Mutant loads from the 8 carriers applying for MELAS PND/PGD (family 1 to 8, Table 3 1) ranged from 5 to 40%, 8 to 50%, and 15 to 80%, in leucocytes, oral mucosa cells, 4 and urinary tract cells, respectively. The lowest and highest values were consistently 5 found in white blood cells and urinary tract cells, respectively, as expected from 6 previous reports [START_REF] Chinnery | Molecular pathology of 26 MELAS and MERRF. The relationship between mutation load and clinical 1 phenotypes[END_REF][START_REF] Chinnery | Nonrandom tissue distribution of mutant mtDNA[END_REF]Frederiksen et al., 7 2006;[START_REF] Whittaker | Urine heteroplasmy is the best 16 predictor of clinical outcome in the m.3243A>G mtDNA mutation[END_REF]. 8

Mutant load assessment in single lymphocytes from 5 carriers showed large 9 intercellular variations, ranging from 0 to 81%, 0 to 81%, 0 to 95%, 0 to 21%, and 0 to 10 100 in patients 1, 2, 3, 5, and 14, respectively. When pooling values from all cells 11 analyzed for a given individual, average mutant load was very close to that achieved 12 from the overall lymphocyte extract recovered from a 10ml blood sample. 13

Comparison of the mean heteroplasmy rate vs one standard deviation of the 14 heteroplasmy distribution at the single-cell level for each of the five lymphocyte pools 15 (16.2±8.6%) showed a 0.92 correlation coefficient (p<0.05) with a positive slope 16

(1.14±0.3) (Table 2 and Supp. Figure S2). 17 Mutant loads were assessed in extraembryonic or embryonic tissues collected 1 between 10GW to term, in 12 fetuses from 7 carriers (families 2 to 8, Table 4). 2

One fetus did not carry the mutation in his two tested tissues (amniocytes at 3 16 GW, and cord blood at birth, fetus 4). As observed in early embryos, no fetus was 4 homoplasmic for the mutant. The mutation load ranged from 4% to 79% in the 11 5 carriers. There was no substantial variation in mutant loads assessed at various 6 stages of pregnancies until birth (Table 4). When excluding data from extra-7 embryonic tissues (10GW trophoblast or at term placenta), m.3243A>G mutant loads 8

were identical in all tested tissues from a given fetus [mean±SD: 74.6±0.7% (3 9 tissues), 42±0.8% (5 tissues), 71±2% (5 tissues), and 78±0.9% (7 tissues) for fetuses 10 2a, 2b, 5b and 7a, respectively]. When placenta and other tissues were available at 11 the same term of gestation (3 fetuses), values were similar in two fetuses (74.6 vs 12 74% and 78 vs 78% for fetus 2a and 7a, respectively), while fetus' 2b mutant load 13 was higher in placenta than in other tissues (57±2% vs 42±2%, respectively). Multiple 14 samples in various parts of placenta, aimed at testing the mutant load distribution, 15 did not show any substantial variation in 2, 6, 10, and 18 various loci of fetuses' 2a, 16 2b, 2c, and 3 placentas, respectively (Table 4), irrespective of the gestation stage (12 17 GW or at birth). 18

Single trophoblastic cells (n=83) and amniocytes (n=64) were isolated from 6 19 fetuses to investigate the intercellular mutant load variation (Figure 2). Simultaneous 20 analyses of parents' and fetal cell DNAs using a polymorphic marker proved the fetal 21 origin and the absence of contamination by maternal DNA of the analyzed cells (not 22 shown). Large intercellular variations were found both in trophoblastic cells (range: 7-23 21%, 6-38%, 23-55%, 23-70%, and 64-100%, for fetuses 5a, 6, 12b, 8, and 2a, 24 respectively), and amniocytes, either at 14 GW (range : 10-23%, 16-42%, and 52-25 94%, for fetuses 12b, 6 and 5b, respectively), or at 30 GW (range : 9-17% and 5-28% 1 in fetuses 5a and 12b, respectively). When pooling values from all cells analyzed for 2 a given cell type in a given fetus, the average mutant loads were very close to those 3 achieved from overall chorionic villi sampling (CVS) and amniotic fluid sample (AFS ) 4 mutant loads (variation<10%, Figure 2). 5

Comparison of the mean heteroplasmy rate vs one standard deviation of the 6 heteroplasmy distribution at the single-cell level for each of the ten fetal cell pools 7 (34.3±23%) showed a 0.81 correlation coefficient (p<0.01) with a positive slope 8 (0.13±0.03) (Table 2 and Supp. Figure S2). 9 10

Predictive value of a prenatal mutant load for the postnatal outcome 11

Assessment of the predictive value of a prenatal mutant load for the disease severity 12 in the postnatal period is of crucial importance. A follow-up of our small cohort of 13 patients born after PGD (n=1) or PND (n=9) provided a preliminary indication, even 14 though for ethical reasons we could not check for the presence of the mutation in 15 these asymptomatic children. All these children, harbouring mutant loads between 0 16 and 35% in prenatal period, are indeed healthy at 1 month to 5 years of age (Table 17 4). [START_REF] Inoue | 5 Generation of mice with mitochondrial dysfunction by introducing mouse 6 mtDNA carrying a deletion into zygotes[END_REF][START_REF] Fan | A mouse model of mitochondrial disease 4 reveals germline selection against severe mtDNA mutations[END_REF]. We used this approach to study the segregation 1 of m.3243A>G mtDNA molecules during human in utero development, thanks to our 2 PND/PGD activities, offered to carriers of this mutation. 3

These data rely on a robust, specific and sensitive assay we devised for the 4 quantification of the m.3243A>G MELAS mutation in tissues and single cells. 5

While last-cycle labeling PCR indeed is a well-established and agreed-upon 6 standard protocol for the quantification of mtDNA heteroplasmy, a major concern with 7 this approach is the need to add the labeled primer prior to the last PCR cycle. This 8 process indeed runs a significant risk of contamination of both the endogenous PCR 9 product by exogenous DNA, and of the laboratory dedicated to single cell analyses 10 by the endogenous PCR product. Such a risk, being a minor one when amplifying a 11 large amount of DNA, becomes a major hurdle to the use of this method, when 12 applied to single cell analysis. 13 We ascertained the reliability of our test over a wide range of mtDNA copies, from 14

Analysis in Day 3-Day 5 embryos 22

To our knowledge, data on the presence of the m.3243A>G mutation at the Day 3 23 stage of embryonic development have not been reported so far. This mutation was 24 found in 32 of 38 embryos (84%) from 2 unrelated carriers. This transmission rate is 25 embryo where both tissues were available (Table 3), additionally indicating that the 20 mtDNA molecule segregation is not skewed across embryonic and extraembryonic 21 cells (trophectoderm) at this developmental stage. Finally, inter blastomere stability of 22 the mutant load suggests that embryonic heteroplasmy determined from a single cell 23 analysis provides an accurate estimate of the whole embryo mutant load, as 24 The m.3243A>G mutant load was found to be highly heterogeneous among 3 various embryos from a given individual, ranging from 18 to 50% and from 0 to 77% 4 in patients' 1 and 2 embryos, respectively. These values on "early" embryos are in 5 agreement again with those on primary oocytes from a m.3243A>G carrier, the 6 mutant load of which ranged from 1 to 50% [START_REF] Brown | Random 13 genetic drift determines the level of mutant mtDNA in human primary oocytes[END_REF]. Two conclusions 7 can be derived from these observations. 8

The first conclusion concerns the mode of transmission of m.3243A>G from 9 the mother to her progeny. The distribution of the mutation level values among 10 Patient 2's embryos was consistent with the Kimura distribution calculated from the 11 neutral drift theory. Furthermore, an equal number of her embryos were found to 12 harbour a mutation level greater than the mean value (18 embryos) and less than the 13 mean (17 embryos). Finally, the mean level of heteroplasmy amongst all embryos 14 was very close to the heteroplasmy level seen in the mother (oral mucosal cells and 15 blood, patients 1 and 2). These data argue for random genetic drift as the 16 mechanism governing mtDNA segregation during oogenesis. This observation in 17 humans is in full agreement with the results of a study of heteroplasmy distribution in 18 the progeny of mice carrying apparently neutral BALB/c and NZB mtDNA sequence 19 variants [START_REF] Jenuth | Random genetic drift in the 8 female germline explains the rapid segregation of mammalian mitochondrial 9 DNA[END_REF]. Assuming that blastomere analysis provides a direct 20 insight on mature oocyte mutant load, our data, gathered with those of Brown et al.,21 suggest that mtDNA segregation is governed by a random genetic drift mechanism 22 over the whole oogenesis period. 23

The second conclusion regards the size of the bottleneck operating for 24 m.3243A>G during oogenesis (table 5). The embryos from patient 2 had a larger 25 parameter, than the oocytes from the individual in Brown's study [START_REF] Brown | Random 13 genetic drift determines the level of mutant mtDNA in human primary oocytes[END_REF]. 2

The confidence intervals did not quite overlap, indicating that there is a real 3 difference between the bottleneck sizes in these two individuals, though this 4 difference is not large. These data therefore suggest that some individual-dependent 5 parameter modulates the bottleneck size for a given mtDNA mutation. 6 7

Analysis in 10 -37 GW fetuses 8

Very few data are available on m.3243A>G segregation during fetal development so 9 far. We looked for this mutation in 12 fetuses from 8 carriers, and aggregated these 10 data with those that we (patients 12 and 13, Table 4 carriers passed on the mutation to 49 of their 58 offspring (84%, Tables 3 and4). 15

This transmission risk fits with the MELAS transmission rate calculated from the 16 mutant load determined in buccal mucosa samples from carriers' offspring in the 17 postnatal period [START_REF] Uusimaa | Prevalence, 2 segregation, and phenotype of the mitochondrial DNA 3243A>G mutation in 3 children[END_REF]. It can not be ascertained whether the 3 18 fetuses shown to be mutation-free at 10GW in the current study were mutation-free 19 embryos initially or alternatively resulted from subsequent loss of mutant mtDNA 20 molecules they carried at early embryonic stage. All fetuses but one (fetus 2c) were 21 indeed conceived out of a PGD procedure, and mutant load values at the Day 3 22 stage were therefore not available. Our data however argue for mutant load stability 23 throughout the entire period of embryofetal development. In fetus 2c, loads in 14GW 24 amniocytes and in term cord blood were indeed 4 and 5%, respectively, in agreement 25 with those assessed at early embryonic stage (embryos 7 and 11 from patient 2, 1 carrying 5 and 14%, respectively, only one of whom, -fetus 2c-, developed). We 2 similarly did not find any temporal variation of the MELAS mutant load in multiple 3 samples taken within 10 to 39 GW (Table 4 A and B). 4

Study of various tissues from six 12-25GW fetuses (2a, 2b, 5b, 7a, 9, 10) 5

showed a tight intertissue stability in each of them. Mutant load assessed in extra-6 embryonic (placenta) and embryonic tissues collected at the same gestation term 7 from 4 fetuses, was found to be similar in 3 of them (2a, 7a, and 9), being higher in 8 trophoblast (57 vs 42%) from the remaining one (fetus 2b). Discrepancy between 9 trophoblast and non trophoblastic fetal tissues did not seem to result from an 10 ascertainment bias secondary to loco-regional mutant load heterogeneity within 11 placenta, recently shown to exist [START_REF] Marchington | Mosaicism for 9 mitochondrial DNA polymorphic variants in placenta has implications for the 10 feasibility of prenatal diagnosis in mtDNA diseases[END_REF]. The heteroplasmy level 12 was indeed highly similar in six separate regions of fetus 2b placenta (Table 4). Thus, 13 this placento-fetal discrepancy might be accounted for by selection of a few cells 14 which will form the first source for the placental membranes in the partitioning 15 blastocyst. This observation additionally emphasizes that analysis of single CVS 16 sample, carried out in a PND frame, may fail to assess the fetal mutant load reliably, 17 as recently reported [START_REF] Marchington | Information for genetic management 14 of mtDNA disease: sampling pathogenic mtDNA mutants in the human 15 germline and in placenta[END_REF]. 18

The observation of tight mutant load stability, both across tissues and with 19 time, in all tested fetuses, suggests that mtDNA segregation is mainly governed by 20 random genetic drift in somatic tissues throughout the entire period of human 21 embryofetal development. Furthermore, as already noted in embryos, none of the 20 22 fetuses analyzed so far (Table 4) was found to carry a heteroplasmy rate exceeding 23 80%, over a wide range of maternal mutant loads (from 5% in WBC to 80% in urinary 24 tract cells). Whether such a 80 % value has a biological significance (i.e., a 25 Since mutations such as m3243A>G exhibit a relatively normal pattern of distribution 2 around the maternal mean, it would be unlikely, given the relatively small sample size 3 and the mean maternal mutant load value in majority below 40 % (in blood) in our 4 series (table 1) to find embryos with greater than 80 % heteroplasmy, even in the 5 absence of selection. 6

We attempted to establish at the single cell level the basis of the mutant load 7 stability observed across fetal tissues. We thus analyzed 147 cells distributed into ten 8 pools of trophoblastic or amniotic cells (7-30 cells per pool) collected in six unrelated 9 fetuses within the 10-30 GW period (Figure 2). Except for one homoplasmic mutant 10 cell (fetus 2a), all cells were heteroplasmic with a mutant load ranging from 5 to 95%. 11

This observation is in line with the few studies available on MELAS single cells 12 collected during the postnatal life, that indeed failed to detect mutant loads over 92% 13 and 98% in lymphocytes [START_REF] Saitoh | Single-cell 14 analysis of mitochondrial DNA in patients and a carrier of the tRNA(Leu)(UUR) 15 gene mutation[END_REF] and muscle fibers (Silvestri et al., 14 2000), respectively. These data argue that, over a critical threshold of heteroplasmy, 15 the resulting impact on respiratory chain function promotes fetal cell death 16 [START_REF] Sasarman | The A3243G tRNALeu(UUR) 17 MELAS mutation causes amino acid misincorporation and a combined 18 respiratory chain assembly defect partially suppressed by overexpression of 19 EFTu and EFG2[END_REF]. 17

When pooling values from all cells belonging to a given pool, mutant load 18 dispersion ranged from 10 to 47% (Figure 2). The frequency distribution of single cell 19 mutant load within each of the ten cell pools corresponded to a binomial distribution, 20 with a median value identical or very close to the mean value of heteroplasmy, in 21 keeping with the random genetic drift mechanism. We subsequently looked for an 22 optional relationship between a mutation rate at the tissue (chorionic villi or 23 amniocytes) level and the amplitude of mutant load dispersion at the single cell level 24 in this tissue (Supp. Figure S2). A previous study using adult carrier lymphocytes had 25 indeed suggested that, the higher the proportion of mutated mtDNA molecules, the 1 wider the dispersion of heteroplasmy level [START_REF] Saitoh | Single-cell 14 analysis of mitochondrial DNA in patients and a carrier of the tRNA(Leu)(UUR) 15 gene mutation[END_REF]. Though the number 2 of single cells analyzed was low in each pool, we however assumed that each of the 3 ten cell pools was fairly representative of the whole tissue sample, based on mean 4 mutant load variance consistently less than 10% between the cell pool and the whole 5 sample. It clearly appeared that, the higher the tissue mutant load, the larger the 6 heteroplasmy dispersion in trophoblastic and amniotic cells, even if the low number 7 of analyzed cells precluded any firm conclusion on the biological relevance of this 8 observation. We then attempted to evaluate whether such a hypothetical relationship 9 can be considered in various tissues from m.3243A>G carriers, collected in prenatal 10 and postnatal periods as well. We considered that Saitoh's study was exposed to 11 some bias in heteroplasmy assessment, due to the complex experimental procedure 12 of this study (first PCR, electrophoresis gel extraction of the PCR products, second 13 PCR, PCR products enzymatic digestion, southern blotting, and bioimager analysis). 14

Using DNA from five adult carrier females, we therefore recapitulated Saitoh's study 15 with our own method of mutant load assessment, in an effort to homogenize our 16 data. We thus found a statistically significant correlation between the mean mutant 17 load in each of the five lymphocyte pools and the heteroplasmy distribution at the 18 single cell level. Interestingly, comparison of our data in lymphocytes and fetal cells 19 showed a marked difference between curve slopes for a same range of 20 heteroplasmy, arguing for some tissue-dependence of mtDNA segregation at the cell 21 level (Supp Figure S2). These data contrast with the few available data of the 22 literature, regarding single muscle fibers in adult carriers. The latter indeed showed 23 that, the higher the mean mutant load of a cell pool, the lower the heteroplasmy 24 distribution at the cell level [START_REF] Petruzzella | 1 Extremely high levels of mutant mtDNAs co-localize with cytochrome c 2 oxidase-negative ragged-red fibers in patients harboring a point mutation at nt 3 3243[END_REF] , 2000). However, the range of mutant loads does not overlap between skeletal 1 muscle (60-100%) and the other tissues (5-60%). Thus, assuming that the apparent 2 relationship between mean mutant loads at a whole sample level and mutant load 3 dispersion at the single cell level makes biological sense in all these tissues, it would 4 remain to be seen whether the proposed tissue-dependance of such correlations is 5 valid throughout the 0-100% range of heteroplasmy. 6 7

Comparison of prenatal and postnatal data 8

It is difficult to reconcile the prenatal inter-tissue stability observed in this study, and 9 the tissue-dependance of m.3243A>G mutant loads in adult carriers, who harbour 10 heteroplasmy rates almost constantly higher in skeletal muscle, urinary epithelial 11 tract cells, and hair follicles, than in white blood cells [START_REF] Chinnery | Nonrandom tissue distribution of mutant mtDNA[END_REF]12 Frederiksen et al., 2006;[START_REF] Whittaker | Urine heteroplasmy is the best 16 predictor of clinical outcome in the m.3243A>G mtDNA mutation[END_REF]. 13

This tissue dependance has been suggested to result from a replicative 14 disadvantage of cells harbouring a high mutant load, thus promoting "mutation 15 epuration" from fast regenerating tissues. Should such a biological process operate 16 prenatally, it could be missed out within the "short" course of a pregnancy, by 17 reference to the very slow decrease of mean mutation level in adult white blood cells 18 [START_REF] Rajasimha | Selection against pathogenic 8 mtDNA mutations in a stem cell population leads to the loss of the 3243A-->G 9 mutation in blood[END_REF], thus reflecting the apparent inter-tissue mutant load stability 19 throughout the prenatal period. Alternatively, cell proliferation rate would be identical 20 among all tissues throughout intra-uterine life, and would become tissue dependant 21 from a fairly late postnatal period. A similar value of mutant load (65 to 70%) has 22 indeed been reported in amniocytes at 21 GW, and in peripheral blood and hair 23 follicles sampled in a four-year old carrier [START_REF] Chou | Prenatal 8 diagnosis of a fetus harboring an intermediate load of the A3243G mtDNA 9 mutation in a maternal carrier diagnosed with MELAS syndrome[END_REF]. NARP, highlights striking differences (Figure 3). 5

While all MELAS embryos herein reported, originating from 20 %-mutant load 6 carriers, are mutation-free or heteroplasmic up to 75% with an apparently random 7 distribution of heteroplasmy, the only two reports on m.8993T>G at Day 3-Day 5 8 embryonic stage indicated that the 3 NARP embryos from a 18-% mutant load carrier 9 were either wild-type or mutant homoplasmic [START_REF] Steffann | Analysis 1 of mtDNA variant segregation during early human embryonic development: a 2 tool for successful NARP preimplantation diagnosis[END_REF], the 5 remaining 10 ones carrying low levels of heteroplasmy (4-22%, [START_REF] Tajima | The development 13 of novel quantification assay for mitochondrial DNA heteroplasmy aimed at 14 preimplantation genetic diagnosis of Leigh encephalopathy[END_REF]. Though the 11 low number of embryos analyzed so far precludes drawing firm conclusion on a 12 putative difference of segregation between m.8993T>G and m.3243A>G molecules 13 during oogenesis, these apparently different patterns of segregation are however 14 substantiated by the few available analyses of primary oocytes from carriers of these 15 mutations [START_REF] Blok | Skewed segregation of the mtDNA 5 nt 8993 (T-->G) mutation in human oocytes[END_REF][START_REF] Brown | Random 13 genetic drift determines the level of mutant mtDNA in human primary oocytes[END_REF]. A wild-type (1/7) or mutant 16 homoplasmic state (6/7) was indeed the rule in the 7 NARP primary oocytes from a 17 50%-mutant load carrier, contrasting with the presence of heteroplasmy in 90% of 82 18 primary oocytes in a m.3243A>G carrier with a 8 %-mutant load in WBC. It can be 19 speculated from these data that bottleneck would be of different size between 20 m.3243A>G and m.8993T>G. 21

Whether a purifying selection operates in the human germline against 22 m.8993T>G and m.3243A>G is a matter of debate. Our data in early NARP embryos 23 [START_REF] Steffann | Analysis 1 of mtDNA variant segregation during early human embryonic development: a 2 tool for successful NARP preimplantation diagnosis[END_REF], those from the analysis of primary oocytes in a m8993T>G 24 carrier [START_REF] Blok | Skewed segregation of the mtDNA 5 nt 8993 (T-->G) mutation in human oocytes[END_REF], as well as our previous report on 2 fetuses with a mutant 25 owing to the limited number of data we achieved, all drawn from low (20 %) mutant 5 load carriers. It has however to be emphasized that we never observed any fetus 6 with a mutant load over 80% among the 8 pregnancies from 6 m.3243A>G carriers 7 with a WBC mutant load equal or even higher than 30 % (patients 6-9, 11, 12, table 8 1), These data are difficult to reconcile with those from murine germline segregation 9 studies indicating that mutations in protein coding genes of mtDNA are strongly 10 selected against, whereas mutations affecting tRNA genes (such as m.3243A>G) are 11 largely refractory from such a process [START_REF] Fan | A mouse model of mitochondrial disease 4 reveals germline selection against severe mtDNA mutations[END_REF][START_REF] Stewart | Strong purifying selection in transmission of mammalian mitochondrial 10 DNA[END_REF]. 12 Regarding the somatic tissue development, we previously showed the 13 existence of fetuses carrying m.8993T>G with various degrees of mutant loads, from 14 0% to 100 %, over the 10-GW stage of pregnancy until delivery (Steffann et al., 15 2007). m.8993T>G and m.3243A>G mutant loads remain at a steady-state level with 16 time or across various tissues, thus supporting a random segregation of mutant 17 mtDNA molecules throughout somatic embryo-fetogenesis irrespective of the 18 mutation type. 19

In conclusion, one can speculate that the bottleneck size, and possibly, the 20 mutant load threshold critical for embryo/fetus survival, varies among mtDNA 21 mutations, thus hampering genetic counselling and PND/PGD procedures in 22 mitochondriopathies resulting from "private" mtDNA mutations. To test this 23 speculation, we will need to collect data on mtDNA mutant loads for other rarer 24 mtDNA mutations. Patient numbering refers to Table 1. Individual values i) represent the heteroplasmy level of 2 single blastomeres or of embryos in toto, ii) are expressed as mean ± SD when more than two blastomeres from one embryo were analyzed (number of analyzed blastomeres in brackets). Embryo 10 from family 2 was separated in two parts before PCR analysis, ie, the inner cell mass (I), and the trophectoderm (T). Calculation of "overall mean value" and "overall median value" took into account either the mean of the values achieved trough analysis of all blastomeres when only blastomeres were available, or values from embryos only in other cases. 0 0 13c 0 0 A, Fetuses analyzed in this study. Chorionic villi (CVS) were sampled at 10 GW, and amniotic fluids (AF) at 14 (AF1) and 30 (AF2) GW, respectively. The other tissues were sampled at 12 GW (fetuses 2a and 2b), or at 19, 20, 24, and 25 GW for fetuses 5b, 7, 9, and 10, respectively, and at term for fetuses 2c and 3. Cord blood was sampled at birth for fetuses 2c (38GW), 3 (37GW) and 4 (39 GW). All assays were carried out in triplicate. Values are expressed as mean ±SD. Placenta values measured on

25

 25

  to assess m.3243A>G mutant load at the single cell 1 level. 2

18

 18 This study was approved by the National Ethics Comittee from l'Agencede 19 Biomedecine and all couples gave informed consent for DNA analyses in themselves 20 and their embryos/fetuses. 21 PND and/or PGD analyses were carried out in 8 individuals from 7 unrelated 22 families, heteroplasmic for the MELAS m.3243A>G mutation. The questionable 23 predictive value of a fetal mutant load for the postnatal outcome was emphasized to 24 all of the couples. In such situations, counseling necessarily refered to postnatal 25 to correlate clinical outcome and mutant load[START_REF] Chinnery | Molecular pathology of 26 MELAS and MERRF. The relationship between mutation load and clinical 1 phenotypes[END_REF]; 1 Uusimaa et al., 2007; Whittaker et al., 2009), and to our personal experience with 2 PGD/PND for another mtDNA disorder, ie NARP (Gigarel et al., 2005; Steffann et al., 3 2006; Steffann et al., 2007) and with PND for MELAS

25

 25

25

 25

3 analyses 4

 34 Courtaboeuf, France), and double-distilled water up to a 25µl final volume. PCR 1 program was as described above (20 PCR cycles). 2StatisticalVariance analysis and calculation of inter or intra class correlation coefficients were 5 used to analyze quantitative traits. Qualitative traits were analyzed using à Chi2 test. 6The 95% confidence intervals of the measurements from Patient 2 were made by 7 fitting a Kimura distribution (Wonnapinji et al., 2008) to the 35 mutation level 8 measurements from this patient, and then drawing 10,000 simulated sets of 35 9 measurements from this Kimura distribution to estimate the confidence intervals due 10 to sample size effects (Wonnapinji et al., 2010).

25

 25

18 Analysis in Day 3 -

 183 Day 5 embryos 19Thirty-eight embryos from 2 unrelated heteroplasmic women were analyzed. The 20 m.3243A>G mutant load was assessed in 31 whole embryos and 70 single 21 blastomeres (Table3). Exogenous DNA contamination of the embryonic sample was 22 never detected in any experiment. 23Within the whole cohort of analyzed embryos, six had no detectable mutation 24 (detection threshold: 2%). Taking into account Patient 2 only (35 embryos), the 25 rate was 83%. None of the embryos were homoplasmic mutant. 1Heteroplasmy levels ranged from 5 to 77%, with an apparently homogeneous 2 dispersion of the mutant rates among embryos. The mutant load value was similar in 3 all blastomeres derived from a single embryo (n = 2 to 11) with a 6% maximal 4 interblastomere variability. The intraclass correlation coefficient (ICC) was equal to 5 0.994, consistent with the high level of agreement of intra-embryo measurements, 6and contrasting with the inter-embryo variability. When possible, comparison of 7 heteroplasmy levels between the whole embryo (collected at Day 5) and two of 8 his/her blastomeres (collected at Day 3) showed a variation rate <7% in 13/16 9 embryos, and between 10 and 15% in the remaining ones. 10When considering all embryos from patient 2, the distribution of the mutation 11 level values was consistent with the distribution predicted from neutral drift theory (p-12 value of 0.53 by KS test) comparing the data against the null hypothesis of the 13 Kimura distribution(Wonnapinji et al., 2008). When pooling mutant loads measured 14 in each embryo from a given woman, mean values were 30±15% and 32±23% for 15 patients 1 and 2, respectively. Each of these values was close to the mutant load 16 measured in oral mucosa cell DNA from the corresponding woman (30% and 27% for 17 patients 1 and 2, respectively). 18 Embryo 10 from family 2, harbouring a 13% mutant load at Day 3, was 19 cultured up to the blastocyst stage (Day 5) and separated in 2 parts, namely, the 20 inner cell mass and the trophectoderm. PCR failed to amplify the inner cell mass 21 DNA, while the heteroplasmy level of the trophectoderm was consistent with the level 22 measured in Day 3 blastomeres (15% vs 13%). 23 24 Analysis in fetuses over 10GW 25

18 19 Discussion 20 21

 1920 Analysis of tissues and cells from human embryos and fetuses is currently the only 22 approach to get some insight into processes that govern maternofetal transmission of 23 common mtDNA mutations in humans, owing to the numerous technological 24 difficulties met in attempts at creating animal models of mitochondrial disorders 25

10 2 to 10 6 .

 6 This range encompassed the mean value of mtDNA copies in 15 lymphocytes [around 150 copies, Urata et al 2008; Maagaard et al. 2006] 16 blastomeres [11 000 -449 000 copies, Lin et al 2004] and oocytes [200 000 -17 800 000 copies, Craven et al. 2010]. Our results demonstrated an insensitivity to 18 template input amounts for this particular method, thus validating the results of the 19 experiments carried out on various cells and tissues. 20 21

  a previous report on the presence of m.3243A>G in adult carrier's 1 primary oocytes[START_REF] Brown | Random 13 genetic drift determines the level of mutant mtDNA in human primary oocytes[END_REF], whose 74 of 82 oocytes (90%) were 2 heteroplasmic. A woman carrying the pathogenic mutation may thus have mutation-3 free embryos, advocating PGD as an appropriate procedure for preventing 4 recurrence of affected offspring in carriers, at least in the mutant load range reported 5 in this study. 6Mutant DNA molecules were constantly found to be equally distributed among 7 the various blastomeres of a carrier embryo until Day 5, in agreement with data on 8 mtDNA polymorphisms achieved in mouse[START_REF] Dean | Prospect of preimplantation genetic diagnosis for heritable mitochondrial 1 DNA diseases[END_REF] and human embryos 9 (Marchington et al., 2010), as well as on the m.8993T>G (NARP) mutation in human 10 (Steffann et al., 2006). Single-cell analysis thus reflects the whole embryo mutant 11 load, supporting relevance of the usual PGD procedure for assessment of the overall 12 embryo genotypic status. It is however worth noting some occasional variability (up to 13 15%) in mutant load between single cell measurements and the whole embryo. This 14 discrepancy could have arisen from either a technical artifact (such as partial loss of 15 mitochondrion during whole embryo recovery) or physiological distortion in 16 m.3243A>G segregation from Day 3 (blastomere sampling stage) to Day 5 (whole 17 embryo recovery stage). This last hypothesis is unlikely since mutant load values 18 were very close in Day 5 trophectoderm and Day 3 single-blastomeres in the only 19

  other mtDNA variants (Steffann et al., 2006; Craven et al., 1 2010) 2

  Wonnapinji et al., 2010), and therefore a smaller bottleneck 1

  highly mutated embryos/fetuses) remains speculative. 1

1 (NARP) mutations throughout human embryofetal development 2

 12 Comparison of mtDNA metabolism in m.3243A>G (MELAS) and m.8993T>GComparing mtDNA segregation data throughout human embryofetal development for 3 two different mtDNA mutations, namely, m.3243A>G/ MELAS and m.8993T>G/ 4

  % from 2 m8993T>G carriers with a 30% mutant load in WBC(Steffann 1 et al. 2007), clearly argue against such a negative selection for this mutation in 2 human. As mentioned above, no conclusion can be drawn at this point on the 3 occurrence of a purifying selection against m.3243A>G in the human germ line, 4

25

 25

3 4 10 Web resources 11 12Figure 1 . 8 Figure 2 . 16 17Figure 3 .Supplementary Figure 1 .Supplementary Figure 2 .

 3101118216312 FIGURES LEGENDS

a

  detection threshold:2%; b PCR failure.

  biopsies, respectively (SD: intersample variation). B, Fetuses reported elsewhere: (9) Matthews et al., 1995, (10) Cardaioli et al., 2000, (11)Chou et al., 2004, (12, 13) our group[START_REF] Bouchet | Prenatal diagnosis of myopathy, encephalopathy, lactic acidosis, 9 and stroke-like syndrome: contribution to understanding mitochondrial DNA 10 segregation during human embryofetal development[END_REF]. In boldface: patients born after PGD or PND.

  , Bouchet et al., 2006) and other 11 groups (patients 9 to 11, Table 4, Matthews et al., 1994; Cardaioli et al., 2000; Chou 12 et al., 2004) had previously published. Maternal m.3243A>G was passed on to 17 of 13

	14	20 fetuses (85% transmission rate). Taking embryonic and fetal data together,

  ;[START_REF] Tokunaga | 20 Single muscle fiber analysis of mitochondrial myopathy, encephalopathy, lactic 21 acidosis, and stroke-like episodes (MELAS)[END_REF], 1994;Silvestri 25

	et al.
	F o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 2

 2 Chou et al., 2004, (12, 13) our group[START_REF] Bouchet | Prenatal diagnosis of myopathy, encephalopathy, lactic acidosis, 9 and stroke-like syndrome: contribution to understanding mitochondrial DNA 10 segregation during human embryofetal development[END_REF]. WBC, white blood cells; OMC, oral mucosa cells; UTC, urinary tract cells.

	and Figure 2); Black symbols: postnatal cell pools:
	lymphocytes (data from Table 2), muscle fibers (data from Petruzella et al.,
	1994; Tokunaga et al., 1994; Silvestri et al., 2000). Each symbol reflects the analysis
	of one cell pool for one individual.

A, Patients analyzed in this study. B, Patients reported elsewhere: (9) Matthews et al., 1994, (

10

)

Cardaioli et al., 2000 , (11)

Table 2 . m.3243A>G mutant load in single cells of various origins Lymphocytes Syncitiotrophoblasts/amniocytes Muscle fibers a

 2 calculation from data by Tokunaga et al., 1994; Petruzella et al., 1994; Silvestri et al., 2000 1

				Page 44 of 48
	Number of patients	5	5	8
	Number of cell pools	5	10	21
	Total cell number	84	147	341
	Heteroplasmy level	16.2 + 8 %	34.3 + 13 %	78.5 + 17 %
	(mean + SD)
	Correlation	0.92 (< 0.05)	0.81 (<0.05)	0.84 (<0.01)
	coefficient (p)
	Slope + SD	1.14 + 0.28	0.13 + 0.03	-0.38 + 0.05

a

Table 3 . m3243A>G mutant load in preimplantation embryos

 3

	Patient	Embryo	Single blastomere	Overall embryo Median Mean (±SD)
			Blastomere 1 Blastomere 2
	1	1			18±1 (n=6)
		2			23±2 (n=5)	23%	30±15%
		3			50±3 (n=3)
	2	1	0 a	0
		2	0	0
		3	0	0	0
		4			0
		5			0
		6			0
		7	5	5
		8	7	8
		9	12	14
		10	13	13	I b T: 15
		11	13	16
		12	17	22	15
		13	14	15	16±2 (n=11)
		14	a	25	23
		15	27	27	23
		16			28
		17	15	21	30
		18	40	41	37	37%	32±23%
		19			42
		20			42
		21			43
		22			47
		23	51	55	48
		24			50
		25			50
		26	39	40	50
		27	54	55	50
		28			55
		29			60
		30	55	55	62
		31	63	66	63
		32	51	54	66
		33	67	68	74
		34	72	77
		35	69	71	75

Table 4 .

 4 m.3243A>G load in extra-embryonic and embryo-fetal tissues

		Patient Fetus CVS	AF1 AF2 Muscle Liver Heart Skin Kidney Lung Gut Brain	Optic nerve	Thymus	Umbilical cord	Placenta	Cord blood
	A	2	2a	77±3		75±1.5		74±3.2		74±0	73, 75 a
			2b	59±6		42±1		41±2	42±2	43±2 43±0.3	57±2 b
			2c		4±0						15±5 c	5±1
		3	3	17±3 16±0.5						22±6 d	16±0
		4	4		0						0
		5	5a	20±0.5 15±3 15±2
			5b		70±1		72±2		73±0	68±2	72±1
			5c	4±0.6
		6	6	32±1	28±1
		7	7a			79±0 78±0.6 77±0.9 78±0.3 79±0 79±0.3	78±1.7	78±1.7
			7b	79±2
		8	8	49±0.7
	B	9	9			55±1	52±4	54±3	51±3	56±5	53±4 52±3 51±3	55±3
		10	10			50	50				50
		11	11			67
		12	12a	35	33	29
			12b	31	23	26
		13	13a	60	63
			13b

Table 5 . Comparative analysis of mutation levels in preimplantation embryos from patient 2 and primary oocytes from a published report Patient 2 Brown et al., 2001

 5

	Sample size (n)	35		82
	Mean mutation level (p 0 )	33.7% [25.8% -42.1%]	12.6% [10% -15%]
	Mutation level variance (V)	0.06	[0.037 -0.086]	0.014 [0.009 -0.020]
	Normalized variance (V/(p 0 (1-	0.27	[0.18 -0.38]	0.13	[0.09 -0.17]
	p 0 ))
	Bottleneck parameter (b)	0.72

John Wiley & Sons, Inc. Human Mutation


Dr Carolina Garcia-Vidal

Susana Padrones

; Carolina

Silvia Fernández-Serrano

Ana Fernández

Cristina Masuet

Jordi Carratalà

Mercè Coromines

Carmen Ardanuy

Francesc Gudiol

Frederic Manresa

Jordi Dorca

Professor Nele Jung

Carolina Sincerely

Garcia-Vidal

Impact of antibiotic therapy on systemic cytokine expression in pneumococcal pneumonia

Keywords: pneumococcal pneumonia, cytokines, antibiotic therapy, IL-6, outcome pneumococcal pneumonia, cytokines, antibiotic therapy, IL-6, outcome

Purpose: The aim of this study was to compare the evolution of systemic cytokine levels over time in patients with pneumococcal pneumonia treated either with ß-lactam monotherapy or with combination therapy (ß-lactam plus fluoroquinolone). Methods: Prospective observational study of hospitalized non-immunocompromised adults with PP. Concentrations of IL-6, IL-8, IL-10 and TNF-α were determined on days 0, 1, 2, 3, 5, and 7. Patients on ß-lactam monotherapy were compared with those receiving combination therapy. Results: Fifty-two patients were enrolled in the study. Concentrations of IL-6, IL-8, and IL-10 decreased rapidly in the first days after admission, in accordance with the mean time to defervescence. High levels of IL-6 were found in patients with the worst outcomes, measured by the need for intensive care unit admission and mortality. No major differences in demographic or clinical characteristics or severity of disease were found between patients treated with ß-lactam monotherapy or combination therapy. IL-6 levels fell more rapidly in patients with combination therapy in the first 48 hours (p=0.016). Conclusions: Our data suggest that systemic expression of IL-6 production in patients with PP is correlated with prognosis. Initial combination antibiotic therapy produces a faster decrease in this cytokine in the first 48 hours.

INTRODUCTION

Streptococcus pneumoniae remains a major cause of disease worldwide [START_REF] Ardanuy | Epidemiology of invasive pneumococcal disease among adult patients in Barcelona before and after pediatric 7valent pneumococcal conjugate vaccine introduction 1997-2007[END_REF]. Among pneumonia pathogens, it is the most common cause of hospitalization in adults and the most frequent cause of death [START_REF] Barlett | Community-acquired pneumonia[END_REF][START_REF] Rosón | Etiology, reasons for hospitalization, risk classes and outcomes of patients with community-acquired pneumonia hospitalized on the basis of conventional admission criteria[END_REF]. Despite improvements in etiologic diagnosis, effective antibiotic therapy and advances in supportive care, the morbidity and mortality rates associated with pneumococcal pneumonia (PP) remain high. Case-fatality rates for bacteremic pneumococcal pneumonia range between 7 and 35% [START_REF] Feikin | Mortality from invasive pneumococcal pneumonia in the era of antibiotic resistance, 1995-1997[END_REF].

A recent study [START_REF] Garcia-Vidal | Early mortality in patients with community-acquired pneumonia: causes, and risk factors[END_REF] of the factors associated with early death in patients with communityacquired pneumonia (CAP) reinforces the classical concept that some deaths are closely related to inadequate host response [START_REF] Austrian | Pneumococcal bacteremia with especial reference to bacteremic pneumococcal pneumonia[END_REF]. Excessive cytokine response in patients with severe CAP has been linked with deleterious effects and poor prognosis [START_REF] Antunes | Systemic cytokine levels in community-acquired pneumonia and their association with disease severity[END_REF][START_REF] Calbo | Systemic expression of cytokine production in patients with severe pneumococcal pneumonia: effects of treatment with a -lactam versus a fluoroquinolone[END_REF][START_REF] Fernández-Serrano | Molecular inflammatory responses measured in blood of patients with severe community-acquired pneumonia[END_REF][START_REF] Kellum | Understanding the inflammatory cytokine response in pneumonia and sepsis: results of the Genetic and Inflammatory markers of sepsis (GenIMS) study[END_REF][START_REF] Menendez | Biomarkers improve mortality prediction by prognostic scales in community-acquired pneumonia[END_REF][START_REF] Monton | Cytokine expression in severe pneumonia: a bronchoalveolar lavage study[END_REF][START_REF] Örtqvist | Diagnostic and prognostic value of Interleukin-6 and C-reactive protein in community-acquired pneumonia[END_REF][START_REF] Xu | Modulation of the inflammatory response to Streptococcus pneumoniae in a model of acute lung tissue infection[END_REF].

However, most studies included populations that were heterogeneous in terms of patients, etiologies, and treatment. Significantly, specific studies of the role of cytokine response for predicting poor outcomes in patients with PP are scarce. In recent years, the modulation of the inflammatory response has emerged as a promising concept for improving the outcomes of CAP. Although it has been suggested that different antibiotic classes may have different effects on the systemic expression of cytokine production [START_REF] Demartini | Effect of multiple doses of clarithromycin and amoxicillin on IL-6, IFNgamma and IL-10 plasma levels in patients with community-acquired pneumonia[END_REF][START_REF] Choi | Effect of moxifloxacin on production of proinflammatory cytokines from human peripheral blood mononuclear cells[END_REF][START_REF] Dalhoff | Immunomodulatory effect of quinolones[END_REF], information addressing this issue in PP is lacking.

We carried out a prospective study in order to (i) analyze the relationship between systemic expression of cytokine production and outcomes in patients with PP and (ii) compare the evolution of systemic cytokine levels over time in patients treated either with -lactam monotherapy or with combination therapy (-lactam plus fluoroquinolone).

MATERIAL AND METHODS

Study subjects and study design. The study was carried out in a 900-bed university hospital for adults in Barcelona, Spain. The hospital serves an area of 1,100,000 inhabitants and admits approximately 24,000 patients per year. All nonimmunocompromised patients with PP who were admitted to the hospital from January 2005 through December 2005 were prospectively recruited and followed up. Patients with neutropenia, HIV infection or transplantation were not included. Concentrations of circulating cytokines were determined for all patients. To assess the effects of treatment on the systemic expression of cytokine production, patients were divided into two groups: those initially treated only with -lactams (-lactam group), and those initially treated with combination therapy including -lactams plus fluoroquinolone (combination therapy group). This prospective longitudinal observational study was approved by the Ethical Committee of our Institution.

Clinical evaluation and follow-up. At the initial visit, and before starting empirical antibiotic therapy, patients underwent a complete clinical history, physical examination and laboratory testing. Microbiological studies included two sets of blood cultures and sputum Gram stain and culture when available. Urinary antigen detection for S. pneumoniae was performed if indicated by the attending physician. Antimicrobial susceptibility was tested by the microdilution method, following the Clinical Laboratory Standard Institute methods and criteria [START_REF]Performance standards for antimicrobial susceptibility testing: 19th informational supplement[END_REF][START_REF]Methods for dilution antimicrobial susceptibility test for bacteria that growth aerobically; approved standard: 7th edition[END_REF]. Empiric antibiotic therapy was administered according to the hospital's guidelines, which recommend the administration of a ß-lactam agent (ceftriaxone or amoxicillinclavulanate) with or without a fluoroquinolone (levofloxacin). Combination therapy was recommended for patients with clinical suspicion of Legionella or an atypical pathogen, or in case of severe CAP in the absence of a demonstrative sputum Gram stain.

Levofloxacin monotherapy was allowed for selected cases (i.e., those patients with allergy to ß-lactam agents and no prior quinolone use).

Patients were seen daily during their hospital stay by one or more of the investigators who provided medical advice when requested and recorded demographic characteristics, underlying disease, clinical features, vaccination status, causative agents, therapy, and outcomes in a computer-assisted protocol.

Definitions. PP was diagnosed in patients with signs and symptoms of an acute-onset lower respiratory tract infection, a new infiltrate on chest radiograph, and one or more cultures positive for S. pneumoniae obtained from blood, normally sterile fluids, or sputum and/or a positive test for detection of urinary antigen. Only good quality samples of sputum (<10 squamous epithelial cells and >25 leucocytes per field) were accepted for processing. S. pneumoniae was identified using standard microbiology procedures.

S. pneumoniae antigen in urine was detected by using a rapid immunochromatographic assay (Now TM , Binax, Portland, ME, USA).

Antimicrobial susceptibility was tested by microdilution. S. pneumoniae strains were serotyped. Molecular characterization was performed by pulsed field gel electrophoresis after restriction with Sma(I) and selected strains were analysed by MLST, as previously reported [START_REF] Ardanuy | Epidemiology of invasive pneumococcal disease among adult patients in Barcelona before and after pediatric 7valent pneumococcal conjugate vaccine introduction 1997-2007[END_REF].

The diagnosis of septic shock was based on a systolic blood pressure of less than 90 mmHg and peripheral hypoperfusion with clinical suspicion of uncontrolled infection.

Early death was defined as death due to any cause < 48 hours of hospitalization. Overall mortality was defined as that due to any cause within 30 days of hospitalization. The severity of illness at presentation was quantified using the validated PORT prediction rule for 30-day mortality and medical complications in CAP, as described elsewhere [START_REF] Fine | A prediction rule to identify low-risk patients with community-acquired pneumonia[END_REF].

Collection of blood samples and laboratory processing. For all patients, serial venous blood samples were collected at inclusion, immediately prior to the initiation of antibiotic therapy, and on days 1, 2, 3, 5, and 7. The blood obtained was placed in tubes containing EDTA, immediately centrifugated, and stored at -80ºC. The assays were performed by one of the authors (M.C.), who was blinded to the clinical details of individual patients. The circulating levels of cytokines IL-6, IL-8, IL-10 and TNF- were measured.

IL-6, IL-8, IL-10 and TNF-, concentrations were measured using commercially available kits (GENZYME, Cambridge, Mass). The procedure consisted of a solid-phase chemiluminescent immunometric assay. The standards defined in the operator's manual were applied. The limits of detection were 3 pg/ml for IL-6, 1 pg/ml for IL-8, 4 pg/ml IL-10 and 0.5pg/ml for TNF-.

Statistical analysis.

To analyze the relationship between systemic expression of cytokine production and the severity of PP we compared serial serum cytokine measurements in patients who had severity markers (bacteremia, ICU admission and mortality) with those who did not. A comparison of serial cytokine measurements was made with the Kruskal-Wallis one-way analysis of variance nonparametric test.

To assess the effects of treatment on the systemic expression of cytokine production, we compared the combination therapy and -lactam groups. Patients who initially received other antibiotic treatments were excluded. To detect significant differences between groups we used the chi-square test with continuity correction for categorical variables.

Normally distributed data were compared by using unpaired t tests and the Mann-Whitney U-test was used for analysis of variables with non-normal distribution. Studies evaluating serum concentrations of cytokines over time (the fall-down pattern) were performed using the general linear model for repeated-measures tests, considering both within-subject and between-subject factors (differences attributable to antimicrobial therapy). The contrasts selected were "difference" and "polynomic" for within subject factors. The analysis was adjusted for potential confounding variables (use of corticosteroids or ICU admission). The data analyses were performed with SPSS software version 13.0. In all analyses, we considered P values less than 0.05 to be statistically significant.

RESULTS

Characteristics of patients and evolution of cytokines over time. Fifty-two

hospitalized patients with PP were included. Their demographic characteristics and main clinical features are shown in Table 1. The diagnosis of PP was established with the use of one or more of the following methods: blood culture (21 cases), sputum Gram stain and culture (20 cases), urinary antigen test (17 cases), and transthoracic needle aspiration (2 cases). All S. pneumoniae were susceptible to ciprofloxacin (MIC range 0.5-2 µg/ml) and to levofloxacin (MIC range 0.5-1µg/ml). Using current non-meningeal breakpoints for beta-lactams, all strains were penicillin (MIC range 0.03-2 µg/ml), amoxicillin (MIC range 0.03-2 µg/ml) and cefotaxime (MIC range 0.03-1 µg/ml) susceptible. The most frequent serotypes were 3, 1 and 5, which accounted for 51.4% of strains. These serotypes were related to ST260 and ST180 for serotype 3, ST306 for serotype 1.

Concentrations of all the cytokines studied were detected in peripheral venous blood samples in all patients, although with a wide range of values. Figure 1 shows the evolution of cytokines over time. At admission, IL-6 and IL-8 showed the highest values.

When the variations in concentrations were analyzed over time, all cytokines except TNF-, showed a statistically significant trend towards a rapid decrease after 24 to 48h. TNF- remained basically unmodified throughout the study period.

Clinical outcomes and their relationship with systemic cytokines. The main outcomes of patients are summarized in table 2. Mean time to defervescence was 2.19 days (SD 1.19). After the initial evaluation in the emergency department, 37 (71.2%) patients were admitted to a conventional hospital ward, whereas the other 15 (28.8%) were transferred to an intensive care unit (ICU). The median length of ICU stay was 7 days (range 2-72 days). The early and overall case-fatality rates were 1.9% and 15.4% respectively. The evolution of systemic cytokines concentration over time in relation to bacteremia was determined, as well as ICU admission and mortality. Table 3 shows serum levels of cytokine on days 0, 1, and 2 in relation to these outcomes. In summary, no significant differences in cytokine levels were found in patients with or without bacteremia. IL-6 was significantly higher in patients requiring ICU admission and in patients who died. High levels of IL-8, especially on day 1, were also documented in patients with ICU admission and in those who died.

On day 0, the third quartile for IL-6 initial concentrations identified 83.3% non-survivors and only 16.2% survivors. Thus, at admission, an IL-6 level > 5206 pg/ml predicted mortality with a sensitivity of 100%, a specificity of 79.3%, a positive predictive value of 80.6%, and a negative predictive value of 100%. On day 1, levels of IL-6 > 4097 pg/ml predicted mortality with a sensitivity of 100%, a specificity of 100%, a positive predictive value of 80%, and a negative predictive value of 94.3%.

Effects of antibiotic treatment on cytokine production.

To assess the effects of treatment on systemic cytokine production, we compared 19 patients in the -lactam group (ceftriaxone in 15 cases and amoxicillin-clavulanate in four) with 20 patients in the combination therapy group (ceftriaxone plus levofloxacin in all cases). Thirteen patients (five initially treated with combination therapy [-lactam plus macrolide], four initially treated with linezolid and four initially treated with a single fluoroquinolone treatment were excluded from the analysis. There were no differences in the characteristics of the patients who were included and those who were excluded. Demographic characteristics, the main clinical features and outcomes of patients by treatment group are shown in Table 4. Interestingly, no important differences in demographic characteristics, vaccination status, time from pneumonia onset to inclusion, previous use of steroids, statins, non-steroidal anti-inflammatory drugs, severity of infection, bacteremia and outcomes were found between groups. Chronic heart and cerebrovascular diseases were more frequent in patients in the -lactam group. Conversely, the presence of multilobar infiltrates was more frequent in the combination therapy group.

As shown in figure 2, the IL-6 decrease was more rapid in the combination therapy group, particularly in the first days (p=0.016). TNF- levels were lower in the combination therapy group, but the differences did not reach statistical significance. No differences in the evolution over time of IL-8 were detected. Conversely, levels of anti-inflammatory cytokines (IL-10) remained higher (p<0.001) in the combination therapy group. All these differences remained significant after adjustment for the use of corticosteroids and ICU admission.

DISCUSSION

Previous studies of CAP have noted that most cytokines can be detected in systemic circulation and show a significant pattern of decline in the first hours of treatment. Indeed, all cytokines studied in the present report were detected in venous blood samples in patients with PP at hospital admission. Thus, most patients developed a systemic extension of the initially compartmentalized immune response in the lung. Interestingly, all these cytokines, except for TNF-, declined in the first 48 hours. These decreases correlate clinically with the time to clinical defervescence.

Previous studies have reported that an excess of proinflammatory cytokines is associated with poor prognosis of CAP [START_REF] Antunes | Systemic cytokine levels in community-acquired pneumonia and their association with disease severity[END_REF][START_REF] Calbo | Systemic expression of cytokine production in patients with severe pneumococcal pneumonia: effects of treatment with a -lactam versus a fluoroquinolone[END_REF][START_REF] Fernández-Serrano | Molecular inflammatory responses measured in blood of patients with severe community-acquired pneumonia[END_REF][START_REF] Kellum | Understanding the inflammatory cytokine response in pneumonia and sepsis: results of the Genetic and Inflammatory markers of sepsis (GenIMS) study[END_REF][START_REF] Menendez | Biomarkers improve mortality prediction by prognostic scales in community-acquired pneumonia[END_REF][START_REF] Monton | Cytokine expression in severe pneumonia: a bronchoalveolar lavage study[END_REF][START_REF] Örtqvist | Diagnostic and prognostic value of Interleukin-6 and C-reactive protein in community-acquired pneumonia[END_REF][START_REF] Xu | Modulation of the inflammatory response to Streptococcus pneumoniae in a model of acute lung tissue infection[END_REF]. Our results showed a similar relationship between high levels of cytokines and poor outcomes in the specific population with PP.

We demonstrated that levels of IL-6 on admission, as well as levels of Il-6 and IL-8 in the first 48h, were the best markers for predicting poor outcomes, in agreement with previous studies analyzing any etiology of CAP. A recent study 11 found that the addition of biological markers such as C-reactive protein to severity scoring systems (PSI, CURB-65 and CRB-65) improves the 30-day mortality prediction. Further studies are currently needed to establish the potential role of IL-6 and IL-8 in supplementing prognosis scoring systems in order to achieve a more accurate identification of patients with a greater probability of death.

The most notable finding of this study was the difference in the cytokine profile between patients treated with -lactam monotherapy and those treated with combination therapy.

We found that combination therapy of a -lactam plus fluoroquinolone produced a faster decreased in IL-6 in the initial 48 hours of treatment in patients with PP. Taking into account the relationship between IL-6 and poor prognosis for PP, the modulation of the expression of this cytokine may be a key point for improving patient outcomes. Whether combination therapy can improve outcomes in patients with CAP is a controversial issue [START_REF] Waterer | Monotherapy may be suboptimal for severe bacteremic pneumococcal pneumonia[END_REF][START_REF] Martinez | Addition of a macrolide to a betalactam-based empirical antibiotic regimen is associated with lower in-hospital mortality for patients with bacteremic pneumococcal pneumonia[END_REF][START_REF] Baddour | Combination therapy lowers mortality among severely ill patients with pneumococcal pneumonia[END_REF][START_REF] Rodriguez | Combination antibiotic therapy improves survival in patients with community-acquired pneumonia and shock[END_REF][START_REF] Harbarth | Lack of effect of combination antibiotic therapy on mortality in patients with pneumococcal sepsis[END_REF][START_REF] Aspa | Impact of initial antibiotic choice on mortality from pneumococcal pneumonia[END_REF]. Moreover, most studies have used the combination of -lactam plus macrolide, not -lactam plus fluoroquinolone. A possible explanation for the differences observed in the pattern of systemic cytokine production over time is that -lactam cell wall activity causes the release of cell wall components which act as potent inflammatory inducers [START_REF] Heumann | Gram-positive cell wall stimulates synthesis of tumor necrosis factor alpha and interleukin 6 by human monocytes[END_REF][START_REF] Tomasz | The nature of cell wall derived inflammatory components of pneumococci[END_REF][START_REF] Tuomanen | Induction of pulmonary inflammation by components of the pneumococcal cell surface[END_REF][START_REF] Tuomanen | Immunomodulating effects of antibiotics: literature review[END_REF]. One hypothesis is that combination therapy offers more rapid microbial killing due to the presence of quinolones and hence shortens the exposure of the host to microbial products. Additionally, fluoroquinolones have an intrinsic immunomodulation effect that inhibits the production of certain pro-inflammatory cytokines [START_REF] Demartini | Effect of multiple doses of clarithromycin and amoxicillin on IL-6, IFNgamma and IL-10 plasma levels in patients with community-acquired pneumonia[END_REF][START_REF] Choi | Effect of moxifloxacin on production of proinflammatory cytokines from human peripheral blood mononuclear cells[END_REF][START_REF] Dalhoff | Immunomodulatory effect of quinolones[END_REF][START_REF]Performance standards for antimicrobial susceptibility testing: 19th informational supplement[END_REF][START_REF] Blaine | Modulation of the production of cytokines in titanium-stimulated human peripheral blood monocytes by pharmacological agents: the role of camp-mediated signaling mechanism[END_REF]. The possible beneficial effects of fluoroquinolones on the systemic expression of cytokine response when combined with -lactam therapy in patients with PP have not been previously explored, but our results suggest that their potential anti-inflammatory and immunomodulatory effects persist when combined with -lactam. A previous study [START_REF] Calbo | Systemic expression of cytokine production in patients with severe pneumococcal pneumonia: effects of treatment with a -lactam versus a fluoroquinolone[END_REF] explored the effects of fluoroquinolone monotherapy in modulating the cytokine response in patients with PP, finding that it achieved a faster decrease in serum TNF- production at 120 hours postadmission than -lactam monotherapy.

Our study has limitations that should be acknowledged. First, the study was observational and included a relatively small number of patients. Second, only four patients were treated with fluoroquinolone monotherapy, precluding comparisons in the pattern of systemic expression of cytokine production. Finally, it should be emphasized that our aim was not to establish whether combination therapy of -lactam plus fluoroquinolone has a clear impact on survival in the first hours after admission for PP. Therefore, our results should be interpreted with caution.

We found that IL-6, IL-8, IL-10 were detected in venous blood samples in all patients with PP at hospital admission with a rapid decrease in the first 48h, correlating with clinical defervescence. High levels of IL-6 were found in patients with the worst outcomes. Initial -lactam and fluoroquinolone combination antibiotic therapy produced a faster decrease of this cytokine in the first 48 hours.

Table 1. Demographic characteristics and the main clinical features of patients

Figure 1 .

 1 Figure 1. Sequential cytokine levels in patients with severe pneumococcal pneumonia.

Figure 2 .

 2 Figure 2. Evolution of IL-6 and Il-10 systemic concentrations by treatment group. IL-6

Table 2 . Outcomes of pneumococcal pneumonia of patients hospitalized for community-acquired pneumonia.

 2

	Outcomes

Table 3 . Results of cytokines on days 0 and 1 in the groups with or without bacteremia, with or without ICU admission and deaths or survivors.

 3

		Bacteremia		ICU admission			Mortality
	Cytokine	Median (P25-75) No (n=31)	Median (P25-75) Yes (n=21)	Median (P25-75) p No (n=37)	Median (P25-75) Yes (n=15)	p	Median (P25-75) Survivors (n=46)	Median (P25-75) Deaths (n=8)	p
	Day 0
	IL-6	2945	2700	NS	1890	10335	.028	2190	8878	.020
		(919-4550)	(778-10335)		(850-4488)	(2700-21160)		(835-4492)	(6236-18453)
	IL-8	181	152	NS	165	452	NS	180	148	NS
		(67-307)	(43-975)		(54-256)	(43-1675)		(51-392)	(48-5953)
	IL-10	23	32	NS	20	47	NS	26	91	NS
		(13-84)	(9-60)		(2-63)	(29-207)		(13-64)	(12-259)
	TNF-	3	13	NS	3	12	NS	3	11	NS
		(0.2-9.5)	(0-19)		(0-10)	(2-19)		(0-11)	(4-17)
	Day 1
	IL-6	276	753	NS	237	2700	<.001	276	8992	<.001
		(99-1146)	(234-2375)		(99-848)	(1405-16029)		(107-850)	(2347-21518)
	IL-8	62	39	NS	36	288	<.001	41	217	.050
		(19-229)	(16-164)		(15-76)	(176-837)		(19-165)	(55-1689)
	IL-10	12	10	NS	11	51	NS	11	66	NS
		(3-19)	(14-73)		(4-19)	(5-203)		(3-19)	(3-232)
	TNF-	3	11	NS	3	5	NS	3	6	NS
		(0-5)	(1.2-15)		(0-11)	(0.5-11)		(0-12)	(0-19)
	Day 2
	IL-6	94	87	NS	66	308	<.001	85	2522	.001
		(24-217)	(66-458)		(24-112)	(118-1626)		(30-228)	(1091-5246)
	IL-8	24

Table 4 . Demographic characteristics, main clinical features and outcomes of patients by treatment group.

 4

	Characteristic	-lactams	Combination	p
			group	therapy group
			N= 19		N= 20
	Demographics
	Age, median years (range)	63.3 (18.0)	56.5 (17.0)	.232
	Male, sex	14	73.7	12	60	.501
	Current smoker	6	31.6	9	47.4	.508
	Heavy drinking	3	15.8	1	5	.287
	Vaccination status
	Influenza vaccine (season)	4	21.0	5	25.0	.317
	Pneumococcal vaccination e	1	5.2	3	15.0	.699
	Underlying disease
	COPD f	7	36.8	4	20.0	.160
	Chronic heart disease	9	47.4	0	0	<.001
	Diabetes mellitus	4	21.1	3	15.0	.451
	Cerebrovascular disease	3	15.8	0	0	.036
	Chronic liver disease	1	5.3	0	0	.241
	Chronic renal disease	0	0	0	0	1
	Time from pneumonia onset to inclusion	2.5 (0-15)	3 (1-15)	.975
	Previous use of statins	3	15.8	2	10.0	.661
	Concomitant use of steroids	2	10.5	0	0	.230
	Concomitant use of NSAI* drugs	3	15.8	0	0	.106
	High risk PSI (IV-V)	13	68.4	14	70	1

(0-15)NS

Funding

This study was supported by research grants of Fundación Pi i Sunyer; FUCAP (Fundació Catalana de Pneumologia); REIPI RD06/0008 from the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Spanish Network for the Research in Infectious Diseases; by FIS (070864); and by Institut d´Investigació Biomèdica de Bellvitge (Dr.

Garcia-Vidal).

Transparency declaration

None to declare