Dr Claire De Moreuil
email: claire.demoreuil@chu-brest.fr

MD Marie-Pierre Moineau

MD Maël Padelli

MD Françoise Lede

MD Annabelle Remoue

Christophe Tremouilhac

MD Philippe Merviel

Brigitte Pan Petesch

PhD Brigitte Pan Petesch

MD Emmanuelle Le Moigne

PhD Karine Lacut

PhD Pascale Marcorelles

First trimester serum biomarkers in pregnancies complicated with placental chronic inflammation

Keywords: Villitis, intervillositis, intrauterine growth restriction, angiogenesis, placental growth factor

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Affiliation address:

Département de médecine interne et pneumologie, CHU de Brest, Hôpital La Cavale Blanche, Boulevard Tanguy Prigent, 29609 Brest Cedex, France.

Funding sources:

PlGF tests used in this study were offered by Thermofisher Scientific.

Thermofisher Scientific did not interfer with the study design, with the data analysis and interpretation or with the writing of the manuscript.

Introduction:

Villitis of unknown etiology (VUE) is a common histological lesion reported in five to 15% of third trimester placentas [START_REF] Redline | Villitis of unknown etiology: noninfectious chronic villitis in the placenta[END_REF], firstly described in 1975 as associated with intrauterine growth restriction (IUGR) [START_REF] Altshuler | The human placental villitides: a review of chronic intrauterine infection[END_REF]. VUE is defined by a chronic inflammation with maternal T CD8+ lymphocytes and histiocytes in the placental villi after an exhaustive investigation excluding infectious agents [START_REF] Redline | Villitis of unknown etiology: noninfectious chronic villitis in the placenta[END_REF][START_REF] Kim | Chronic inflammation of the placenta: definition, classification, pathogenesis, and clinical significance[END_REF]. Chronic intervillositis of unknown etiology (CIUE) is a rare lesion (0,6 per 1000 placentas of second and third trimesters) defined by a chronic inflammation with histiocytes and lymphocytes in the intervillous space, after exclusion of infectious agents [START_REF] Boyd | Chronic histiocytic intervillositis: a placental lesion associated with recurrent reproductive loss[END_REF].

VUE and CIUE have been reported in association with recurrent adverse pregnancy outcomes such as early recurrent miscarriage, stillbirth, IUGR, small for gestational age (SGA) and preeclampsia [START_REF] Derricott | Investigating the association of villitis of unknown etiology with stillbirth and fetal growth restriction -a systematic review[END_REF][START_REF] Leftwich | Placental Pathology : Useful for Undiagnosed Small for Gestational Age Neonates?[END_REF]. Pathophysiological findings of VUE and CIUE evoke a common immune origin, i.e. a maternal anti-fetal rejection process [START_REF] Boyd | Chronic histiocytic intervillositis: a placental lesion associated with recurrent reproductive loss[END_REF]. Angiogenic biomarkers have never been studied specifically in plasma of pregnant women affected by VUE or CIUE. Yet, placental growth factor (PlGF) is a validated early biomarker of vasculoplacental disorders related to abnormal placental bed implantation and could be disturbed in adverse pregnancy outcomes associated with exclusive VUE and CIUE associated with VUE [START_REF] Levine | Circulating angiogenic factors and the risk of preeclampsia[END_REF].

In this observational study, we described 78 placentas affected by exclusive VUE or CIUE associated with VUE and corresponding pregnancies and compared them with 75 placentas without chronic inflammation from uncomplicated pregnancies. In addition, we determined if first trimester serum biomarkers (PlGF, PAPP-A, βhCG) could predict adverse pregnancy outcomes associated with VUE and CIUE.

Materials and Methods:

Definition of cases and controls:

Placentas with exclusive VUE or CIUE associated with VUE from pregnancies with a delivery between January 2013 and June 2018 in one of the six maternities of our county participating to "HPP-IPF" (NCT02884804) or "HEMOTHEPP" (NCT02443610) observational studies were qualified as cases. They were selected from the Pathology department files of Brest University Hospital using a computerized database (ADICAP system). Control placentas were selected during the same period among placentas classified as "normal", "with minimal lesions" or "with acute inflammation" from uncomplicated pregnancies (ie without pre-eclampsia, stillbirth, IUGR or SGA).

Multiple gestation pregnancies were excluded.

Histopathological examination of the placentas:

Histopathological examination of the placentas had been performed by two senior perinatal pathologists at Brest University Hospital.

Placentas were fixed in 4% buffered formalin. Standard sampling of three blocks in the central area was performed and slides were Hematoxylin, Eosin and Saffron (HES) stained.

Recorded macroscopic findings corresponded to the following items: placenta weight, fetoplacental weight ratio, placental abruption and other grossly identified lesions.

According to Amsterdam consensus [START_REF] Khong | Sampling and Definitions of Placental Lesions: Amsterdam Placental Workshop Group Consensus Statement[END_REF], a placental weight < 10 th percentile, a thin cord < 10 th percentile, a placental abruption, a macroscopic infarct in a preterm placenta or a nonperipheral infarct > 5% of the chorionic plate in a term placenta were considered as features of maternal vascular malperfusion.

Recorded microscopic findings corresponded to the following items: maternal vascular malperfusion features such as microscopic infarcts, decidual arteriopathy, abnormal villi development and fetal vascular malperfusion features such as avascular villi and stem vessel obliteration.

The following chronic inflammation features were also described: VUE, CIUE, chronic chorioamniotitis and chronic deciduitis. VUE was classified in high or low grade. High grade VUE was defined by the presence of inflammation affecting more than 10 contiguous villi in any focus, with more than one focus required for diagnosis [START_REF] Contro | Chronic intervillositis of the placenta: a systematic review[END_REF]. VUE was also defined according to the location of the inflammatory infiltrate: proximal stem villi and sometimes chorionic plate (i.e. subchorial), distal villi (i.e. parenchymatous), anchoring villi embedded in the basal plate (i.e. basal) [START_REF] Khong | Sampling and Definitions of Placental Lesions: Amsterdam Placental Workshop Group Consensus Statement[END_REF].

Criteria used for CIUE diagnosis were the same as developed by Bos et al [START_REF] Bos | Towards standardized criteria for diagnosing chronic intervillositis of unknown etiology: A systematic review[END_REF]. CIUE could be associated with VUE and was classified as diffuse or focal according to the density of cellular infiltrate [START_REF] Bos | Towards standardized criteria for diagnosing chronic intervillositis of unknown etiology: A systematic review[END_REF][START_REF] Khong | Sampling and Definitions of Placental Lesions: Amsterdam Placental Workshop Group Consensus Statement[END_REF][START_REF] Contro | Chronic intervillositis of the placenta: a systematic review[END_REF][START_REF] Jacques | Chronic intervillositis of the placenta[END_REF].

Data on pregnancies:

Data on pregnancies were recorded from medical files on a computerized database. Baseline maternal characteristics included preconceptional Body Mass Index, medical history, blood and rhesus group, obstetrical history and age at delivery. Pregnancy characteristics included method of conception, medication during pregnancy, gestational diabetes, pre-eclampsia, intrahepatic cholestasis, premature rupture of membrane and term at delivery. The following fetal characteristics were recorded: presence and term at diagnosis of IUGR, SGA, stillbirth, birthweight and sex of the newborn.

Adverse pregnancy outcomes were defined as the occurrence during pregnancy of one of the following complications: stillbirth, IUGR, SGA or pre-eclampsia. Pre-eclampsia was defined according to the American College of Obstetricians and Gynecologists' definition published in 2013 [START_REF]Hypertension in pregnancy. Report of the American College of Obstetricians and Gynecologists' Task Force on Hypertension in Pregnancy[END_REF]. IUGR was defined according to the 2013 French College of Obstetricians and Gynecologists guidelines by an estimated fetal weight below the 10 th percentile using locallyaccepted curve (AUDIPOG) associated with clinical signs of pathology [START_REF] Carbonne | Intra uterine growth retardation: guidelines for clinical practice[END_REF]. SGA was defined by a birthweight below the 10 th percentile.

Biological parameters:

First trimester Down syndrome screening results were collected for each pregnancy.

First trimester pregnancy associated plasma protein-A (PAPP-A) and β-human chorionic gonadotrophin (βhCG) levels had been measured in international unit/liter and converted to multiples of the median (MoM) using the crown-rump length or biparietal diameter measurement when the blood sample was obtained as an estimate of gestational age.

Available blood samples collected at the end of first trimester of pregnancy (between 11 weeks of gestation (WG) and 13+6 WG) for Down syndrome screening stored at -20°C in Biochemistry department of Brest University Hospital were used for PlGF quantification.

Measurements were done with the automated B . R . A . H . M . S KRYPTOR compact PLUS system (B . R . A . H . M . S PlGF plus KRYPTOR: Thermo Fisher Scientific, Hennigsdorf, Berlin) according to the manufacturer's instructions described elsewhere [START_REF] Unterscheider | Optimizing the definition of intrauterine growth restriction: the multicenter prospective PORTO Study[END_REF]. PlGF levels were expressed in pg/ml. We compared our results to norm values of PlGF determined by Verlohren et al [19] between 10 and 14 WG and we determined the number of values < 2,5 th percentile (corresponding to 26,80 pg/ml) in each group.

The institutional review board of Brest University Hospital approved "HPP-IPF" and "HEMOTHEPP" studies. Informed oral consents were obtained from all participants.

Informed written consents were obtained from patients whose blood samples had been collected at the end of first trimester and who had not been enrolled in one of these two studies.

Statistical analysis:

Quantitative variables were summarized using median and interquartile range (IQR).

Categorical variables were summarized as frequency (%) of categories.

Difference between groups for categorical variables were analyzed by the Chi square test (or Fisher's exact test if the number of individuals was < 5). For continuous variables, differences between groups were analyzed by nonparametric tests: the Mann and Whitney U test for comparison between two groups and the Kruskall Wallis test for comparison between more than two groups. The significance level used was p < 0,05.

All statistical analyses were performed with GraphPad Prism V5.

Results:

Cases: 675 placentas with chronic inflammation were identified by interrogation of ADICAP system between January 2013 and June 2018 (figure 1). At the same period, 6685 placentas had been examined. Among these 675 placentas, after excluding placentas from multiple gestation pregnancies, placentas with inflammation of infectious etiology and placentas without first trimester Down syndrome screening results, 81 placentas with VUE and/or CIUE were included. 3 placentas with exclusive CIUE were finally excluded because this sub-group was too small to be representative. Of the 78 remaining placentas, 40 corresponding patients had a stored serum sample available at first trimester of pregnancy for PlGF quantification. Among these 78 cases, 16 (20,5%) were CIUE associated with VUE, accepting their cooccurrence in a placenta, and 62 (79,5%) were exclusive VUE.

Controls:

2547 placentas labelled "normal" or "with minimal lesions" or "with acute inflammation" were identified at the same period (figure 1). After excluding multiple gestation pregnancies, pregnancies complicated with pre-eclampsia, stillbirth, IUGR or SGA, placentas with chronic inflammation and placentas without first trimester Down syndrome screening results, 75 control placentas were included. Of these, 74 corresponding patients had a stored serum sample available at first trimester of pregnancy for PlGF quantification.

Comparison between cases and controls:

Baseline maternal characteristics were similar in cases and controls (table 1).

In the case group, adverse pregnancy outcomes were frequent: 21,8% IUGR, 30,8% SGA and 10,3% pre-eclampsia (table 2).

Cases had less gestational diabetes (12,8% vs 26,7%, p = 0,04) than controls. Median term at delivery was similar in cases and controls (39 and 38 WG). 3). Case placentas showed more excessive fibrin deposition (87,2% vs 61,3%, p = 0,0004), more maternal vascular malperfusion features (79,5% vs 22,7%, p < 0,0001) and more fetal vascular malperfusion features (33,3% vs 12%, p = 0,002).

Median PAPP-A levels were similar in cases and in controls (0,83 MoM [IQR 0,55-1,26] vs 1,02 MoM [IQR 0,70-1,29], p = 0,09). Cases had lower βhCG levels than controls (0,74 MoM [IQR 0,53-1,12] vs 1,00 MoM [IQR 0,72-1,53], p = 0,002) (table 4 and figure 2 C).

First trimester PlGF levels were measured in 40 cases and in 74 controls. Median PlGF levels were lower in cases than in controls (29,74 pg/ml [IQR 19,74-36,17] vs 36,37 pg/ml [IQR 27,36-49,13], p = 0,007) (table 4 and figure 2 A).

In the case group, 24 pregnancies complicated with adverse pregnancy outcomes (i.e. 9 IUGR, 13 SGA and 4 pre-eclampsia) had a PlGF measurement at first trimester. 14 among 24 of these pregnancies complicated with adverse pregnancy outcomes (i.e. 58,3%) had a PlGF level < 2,5 th percentile.

Comparison between exclusive VUE and CIUE associated with VUE:

16 CIUE associated with VUE and 62 exclusive VUE were included in this study (table 5).

Baseline maternal characteristics were similar between the CIUE associated with VUE group and the exclusive VUE group.

Adverse pregnancy outcomes rate was similar in the two groups, as well as median term at delivery.

Median newborn birthweight was lower in the exclusive VUE group in comparison with the CIUE associated with VUE group (2580 vs 2805 g, p = 0,04).

Median first trimester PAPP-A and βhCG levels were similar in the two groups (0,85 MoM vs 0,82 MoM, p = 0,8 for PAPP-A and 0,65 MoM vs 0,86 MoM, p = 0,3 for βhCG) (table 6 and figure 2 D).

PlGF levels were lower in CIUE associated with VUE and in exclusive VUE cases compared to controls (respectively 34,05 pg/ml vs 28,35 pg/ml and 36,37 pg/ml, p = 0,01; table 6 and figure 2 B). In the CIUE associated with VUE group, one among five adverse pregnancy outcomes had a PlGF level < 2,5 th percentile, whereas in the exclusive VUE group, 13 among 19 adverse pregnancy outcomes (i.e. 68,4%) had a PlGF level < 2,5 th percentile.

Comment:

Our results confirm that IUGR and SGA are frequent in VUE and CIUE associated with VUE pregnancies. Additionally, we found lower first trimester PlGF levels in exclusive VUE cases than in controls, suggesting that reduced angiogenesis is involved in adverse pregnancy outcomes related to VUE.

We identified 14 studies on VUE or CIUE in PubMed database with a healthy control group, among which only two studies had a design similar to ours [START_REF] Becroft | Placental villitis of unknown origin: epidemiologic associations[END_REF][START_REF] Aviram | Placental aetiologies of foetal growth restriction: clinical and pathological differences[END_REF][START_REF] Gunyeli | Histopathological analysis of the placental lesions in pregnancies complicated with IUGR and stillbirths in comparison with noncomplicated pregnancies[END_REF][START_REF] Althabe | Chronic villitis of unknown aetiology and intrauterine growthretarded infants of normal and low ponderal index[END_REF][START_REF] Salafia | Placental pathology of idiopathic intrauterine growth retardation at term[END_REF][START_REF] Almasry | Structural analysis of human placental stem and terminal villi from normal and idiopathic growth restricted pregnancies[END_REF][START_REF] Kovo | Villitis of unknown etiologyprevalence and clinical associations[END_REF][START_REF] Labarrere | Chronic villitis of unknown etiology and massive chronic intervillositis have similar immune cell composition[END_REF][START_REF] Ananthan | Placental Findings in Singleton Stillbirths: A Case-control Study[END_REF][START_REF] Altemani | Villitis of unknown etiology in placentas of pregnancies with hypertensive disorders and of small-for-gestational-age infants[END_REF][START_REF] Nordenvall | Placental villitis and intrauterine growth retardation in a Swedish population[END_REF][START_REF] Labarrere | Chronic villitis of unknown etiology and maternal arterial lesions in preeclamptic pregnancies[END_REF][START_REF] Labarrere | Chronic villitis of unknown aetiology in placentae of idiopathic small for gestational age infants[END_REF][START_REF] Iskender-Mazman | Placental findings of IUGR and non-IUGR[END_REF]. A high prevalence of autoimmune diseases (systemic lupus erythematous, antiphospholipid syndrome, Sjögren's syndrome…) was described among patients with a history of CIUE [START_REF] Mekinian | Chronic histiocytic intervillositis: outcome, associated diseases and treatment in a multicenter prospective study[END_REF], what we did not confirm.

We found similar adverse pregnancy outcomes rates in exclusive VUE cases and in CIUE associated with VUE cases. Only two other studies compared obstetrical prognosis between VUE and CIUE. Nowak et al found more IUGR in the CIUE group in comparison with VUE and combined VUE-CIUE [START_REF] Nowak | Perinatal prognosis of pregnancies complicated by placental chronic villitis or intervillositis of unknown etiology and combined lesions: About a series of 178 cases[END_REF] whereas Labarrere et al found no difference in terms of SGA and pre-eclampsia between VUE and CIUE [START_REF] Labarrere | Chronic villitis of unknown etiology and massive chronic intervillositis have similar immune cell composition[END_REF].

VUE and CIUE share a similar immune composition and a common pathophysiology. Lee et al considered VUE, chronic chorioamniotitis and chronic deciduitis as one global and unique placental manifestation of the maternal anti-fetal rejection process, characterized by a fetal systemic inflammation with increased fetal plasma angiostatic CXC motif ligand chemokine 10 (CXCL10) called "FIRS type 2" [START_REF] Lee | Characterization of the fetal blood transcriptome and proteome in maternal anti-fetal rejection: evidence of a distinct and novel type of human fetal systemic inflammatory response[END_REF]. This chemokine could be the Ariane thread connecting chronic inflammation, growth restriction and reduced angiogenesis suspected in VUE [START_REF] Romagnani | CXC chemokines: the regulatory link between inflammation and angiogenesis[END_REF].

A high rate of maternal vascular malperfusion features in placentas of IUGR and SGA suggests VUE and CIUE associated with VUE generate placental underperfusion. This significant pathophysiological association had already been reported by Salafia et al in a study investigating preterm pre-eclampsia [START_REF] Salafia | Placental pathologic features of preterm preeclampsia[END_REF].

To our knowledge, this is the first study evaluating the relevance of angiogenic biomarkers in prediction of adverse pregnancy outcomes in the specific population of VUE and CIUE associated with VUE pregnancies. Our study design aimed at finding a first trimester predictive biomarker of adverse pregnancy outcomes associated with exclusive VUE and CIUE associated with VUE. We documented lower βhCG levels at the end of first trimester in cases compared to controls. Low first trimester βhCG levels have already been described in association with IUGR [START_REF] Smith | Early pregnancy levels of pregnancy-associated plasma protein a and the risk of intrauterine growth restriction, premature birth, preeclampsia, and stillbirth[END_REF] and pregnancy loss before 24 WG [START_REF] Dugoff | Firsttrimester maternal serum PAPP-A and free-beta subunit human chorionic gonadotropin concentrations and nuchal translucency are associated with obstetric complications: a population-based screening study (the FASTER Trial)[END_REF]. Nevertheless, elevated first trimester βhCG levels have been associated with vasculoplacental disorders such as preeclampsia, IUGR and gestational diabetes [START_REF] Merviel | Correlations between serum assays of human chorionic gonadotrophin (hCG) and human placental lactogen (hPL) and pre-eclampsia or intrauterine growth restriction (IUGR) among nulliparas younger than 38 years[END_REF], which makes our results difficult to interpret.

We found lower first trimester PlGF levels in cases compared to controls. Levine et al identified that PlGF levels were lowered from 13 WG in women affected by pre-eclampsia and even lower when pre-eclampsia was associated with IUGR [START_REF] Levine | Circulating angiogenic factors and the risk of preeclampsia[END_REF]. Poon LC et al added that PlGF was also lowered in SGA cases and that PlGF could be used in combination with PAPP-A, uterine artery pulsatility index and maternal factors to predict SGA at first trimester [START_REF] Poon | Maternal serum placental growth factor (PlGF) in small for gestational age pregnancy at 11(+0) to 13(+6) weeks of gestation[END_REF][START_REF] Poon | Combined screening for preeclampsia and small for gestational age at 11-13 weeks[END_REF].

Our findings are consistent with an implication of early angiogenesis dysregulation in adverse pregnancy outcomes associated with exclusive VUE. On the other hand, CIUE associated with VUE cases seem to be unrelated to reduced angiogenesis, as suggested by the low frequency of very low PlGF values in this group. In exclusive VUE cases, the immunological conflict between mother and fetus might trigger reduced angiogenesis through early and abnormal placental hypoxia, as suggested by the high frequency of maternal vascular malperfusion features observed in placentas from IUGR and SGA. First trimester PlGF quantification in pregnant women with a past history of adverse pregnancy outcome related to exclusive VUE could help to predict recurrence of obstetrical complications. Therefore, a low PlGF level could justify aspirin prophylaxis throughout the ongoing pregnancy.

Our study has some limitations. Due to small-sized groups, we were not able to observe a significant association between maternal clinical characteristics and occurrence of VUE or CIUE associated with VUE. Then, PlGF was measured in only half of the cases. Finally, we tried to have a control group with normal placentas from uncomplicated pregnancies.

Nevertheless, as this study was conducted retrospectively, our controls were not completely healthy, as attested by the high proportion of gestational diabetes.

Our study has some strengths: we described VUE and CIUE associated with VUE but also compared them with controls, trying by this approach to find potential predictors of VUE and CIUE associated with VUE. This last point was rarely reported in previous literature.

In summary, exclusive VUE and CIUE associated with VUE seem similarly associated with maternal vascular malperfusion features in placenta and with IUGR and SGA pregnancies.

First trimester low PlGF levels in cases suggest that reduced angiogenesis is involved in adverse pregnancy outcomes related to VUE.

Legends of tables and figures:

  When compared to control placentas, case placentas were smaller (425 g [IQR 370-480] vs 460 g [IQR 390-523], p = 0,03), in correlation with smaller birthweight (2645 g [IQR 2270-2883] vs 2970 g [IQR 2340-3385]) (table

Figure 1 :Figure 2 :

 12 Figure 1 : flow chart of the study

Table 1 : Comparison of maternal characteristics between cases (CIUE associated with VUE and exclusive VUE) and controlsTable 2 : comparison of pregnancies between cases (CIUE associated with VUE and exclusive VUE) and controlsTable 3 : comparison of histological findings between cases (CIUE associated with VUE and exclusive VUE) and controlsTable 4 : comparison of first trimester biological parameters between cases (CIUE associated with VUE and exclusive VUE) and controlsTable 5 : overall comparison between CIUE associated with VUE, exclusive VUE and controlsTable 6 : comparison of biological parameters between CIUE associated with VUE, exclusive VUE and controls

 123456

Table 1 : Comparison of maternal characteristics between cases (CIUE associated with VUE and exclusive VUE) and controls

 1 Continuous variables are expressed as median and interquartile range and categorical variables are expressed as frequency (percent). Chi square test or Fisher's exact test were used for categorical variables and Mann and Whitney U test was used for continuous variables. CIUE = chronic intervillositis of unknown etiology; VUE = villitis of unknown etiology; BMI = body mass index.

	n = 78 cases	n = 75 controls	p

Table 2 : comparison of pregnancies between cases (CIUE associated with VUE and exclusive VUE) and controls

 2

	n = 78 cases	n = 75 controls	p

30.4-70) Sex Male 31 (39.7%) 38 (50.7%) 0.2 Female 47 (60.3%) 37 (49.3%)

  Continuous variables are expressed as median and interquartile range and categorical variables are expressed as frequency (percent). Chi square test or Fisher's exact test were used for categorical variables and Mann and Whitney U test was used for continuous variables. CIUE = chronic intervillositis of unknown etiology; VUE = villitis of unknown etiology; WG = weeks of gestation; IUGR = intrauterine growth restriction; SGA = small for gestational age.

Table 4 : comparison of first trimester biological parameters between cases (CIUE associated with VUE and exclusive VUE) and controls

 4 Continuous variables are expressed as median and interquartile range. CIUE = chronic intervillositis of unknown etiology; VUE = villitis of unknown etiology; PAPP-A = Pregnancy associated plasma protein-A; βhCG = β-human chorionic gonadotrophin; PlGF = placental growth factor; MoM = multiples of the median; 1 st T = first trimester of pregnancy.

	n = 78 cases	n = 75 controls	p

Table 5 : overall comparison between CIUE associated with VUE, exclusive VUE and controls

 5

	Continuous variables are expressed as median and categorical variables are expressed as frequency (percent). Chi square test or Fisher's
	exact test were used for categorical variables and Kruskall Wallis test was used for continuous variables. CIUE = chronic intervillositis of
	unknown etiology; VUE = villitis of unknown etiology; BMI = body mass index; WG = weeks of gestation; IUGR = intrauterine growth
	restriction; SGA = small for gestational age.

Table 6 : comparison of biological parameters between CIUE associated with VUE, exclusive VUE and controls

 6 Continuous variables are expressed as median and interquartile range. Kruskall Wallis test was used for statistical analysis. CIUE = chronic intervillositis of unknown etiology; VUE = villitis of unknown etiology; PAPP-A = Pregnancy associated plasma protein-A; βhCG = β-human chorionic gonadotrophin; PlGF = placental growth factor; MoM = multiples of the median; 1 st T = first trimester of pregnancy.

	Biological parameters	CIUE associated with VUE N = 16	Exclusive VUE n = 62	Controls n = 75	p
	Median PAPP-A at 1 st T (MoM)	0.85	0.82	1.02	0.2
	Median βHCG at 1 st T (MoM)	0.65	0.86	1.00	0.005
		CIUE associated with VUE N = 16	Exclusive VUE n = 33	Controls n = 74	p
	Median PlGF at 1 st T (pg/ml)	34.05	28.35	36.37	0.01

Acknowledgements:

We thank all members of « HPP-IPF » and « HEMOTHEPP » teams: midwives, obstetricians (especially M. Müller, M. Jacquot, G. Salnelle, M. Collet, C. Bellot and D. Margis), research assistants, nurses, data managers and statisticians. We thank the patients included in these two studies. We also thank the technical staff of Pathology and Biochemistry departments.


Stéphane Péan

A Prepeliţă

Laëtitia Demay
email: laetitia.demay@mnhn.fr

Teodor Obadă

Utilization of mammoth resources and occupation of the Dniester-Prut basin territory: The Upper Palaeolithic site of Valea Morilor (Republic of Moldova)

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Within the East European Plain, the Podolian Upland have been heavily settled by palaeolithic human populations. We focus here about the extra-Carpathian area, particularly the Prut-Dniester basin during Upper Pleniglacial, to better understand Upper Palaeolithic cultures. Moreover there are few sites related to the Last Glacial Maximum. That is why the faunal material from Valea Morilor could provide further information. We study the use of natural resources, in particular of animal origin, to inform the strategies of occupation of the territories, placed in their palaeoecological framework. We use zooarchaeological methods to determine the faunal spectrum, mortality profiles of animals, their anatomical representation in relation with taphonomic processes and palaeoethnography to restore the involvement of human activities.

Archaeological investigations

The site of Valea Morilor from the eponymous lake is located in Chişinău. Rescue excavations were conducted during three campaigns in 2009 and 2010, by S.I. Covalenco, T. Obadӑ and V.A. Burlacu under the direction of O.G. Levitsky [START_REF] Obadӑ | Preliminary results of studies of the Valea Morilor Upper Palaeolithic site (Chişinău, Republic of Moldova): A new camp of mammoth hunters[END_REF]. The excavation n °1 was conducted in three sectors (1A: 495m²; 1B: 230 m²; 1C: 516 m²). Two other smaller excavations, n ° 2 and n ° 3, and a pit survey were respectively carried out on 16 m², 4 m² and 6 m² [START_REF] Obadӑ | Preliminary results of studies of the Valea Morilor Upper Palaeolithic site (Chişinău, Republic of Moldova): A new camp of mammoth hunters[END_REF] (Figure 1).

Geomorphology, stratigraphy and chronology

The site is located near the Durleşti River, which is characterized by meanders, on a terrace made of yellowish-gray clayey silty sands from river water and slope deposits (Obadӑ et al., 2011). Indeed, the site is located in a basin forming a floodplain.

A stratigraphic survey was carried out on the east wall of the pit n°4. It permits to highlight four large sedimentary units (Figure 2). Bone remains, as well as charcoals, hearths and ashy lenses were located between 2.95 and 3.10 meters deep, in clayey sand. Bone clusters are identified as belonging to a single layer ranging from 0.11 to 0.29 m thick. Under these bones loess dolls were found, typical from loessic deposits relied to cold and dry environment. The site is located to Pleistocene loessic deposit area [START_REF] Haase | Loess in Europe -its spatial distribution based on a European Loess Map[END_REF]. Radiometric datings were performed in Groningen on mammoth bones from all three areas (Obadӑ, Van der Plicht, 2010) (Table 1). It seems that the different sectors are relatively contemporary, dated between 21,000 and 20,000 BP.

Palaeoenvironmental data

According to the study about malacofauna (Prepelitsa et al., 2011;[START_REF] Obadӑ | Preliminary results of studies of the Valea Morilor Upper Palaeolithic site (Chişinău, Republic of Moldova): A new camp of mammoth hunters[END_REF], 162 shells of molluscs have been described belonging to four species: Helicopsis striata (Mull.) [78], Pupilla muscorum (L.), Vallonia pulchella (Mull.), Succinea oblonga (Drap.). They are terrestrial molluscs of dry and cold plains. There are no river species or marshes. Only S. oblonga testifies to the proximity of a watercourse. Moreover concerning malacofauna, these remains are little altered and have not been moved, indicating their rapid burial in the subsurface and the very low intensity of the current in the event of flooding (Prepelitsa et al., 2011).

Concerning the small mammals, studied by Markova in [START_REF] Obadӑ | Preliminary results of studies of the Valea Morilor Upper Palaeolithic site (Chişinău, Republic of Moldova): A new camp of mammoth hunters[END_REF], they are represented by: Ochotona pusilla (pika), Spalax zemni (mole rat), Lagurus lagurus (Steppe lemming). These are also typical steppe species.

According to the palaeoenvironmental reconstructions and the first dating, this site is dated to the Last Glacial Maximum.

Archaeological material

Stone artefacts

During the excavations, 72 flint pieces and 7 pebbles (6 sandstone and 1 quartz) were discovered, distributed over the three sectors of Valea Morilor. It is a semi-transparent Cretaceous gray flint found in the Dniester Valley [START_REF] Obadӑ | Upper Palaeolithic Site Valea Morilor from Central Moldova[END_REF]. They are mostly waste size and retouched blades, as well as a scraper and a burin, without any core (Figure 3). There is no taphonomic modifications evidence of stigma associated with sedimentary or hydraulic movements. According to S. Covalenco a cultural attribution, more precisely than Upper Palaeolithic remains difficult for the moment. Microlith proportions, also as the burin prepared on a large massive blade, without gravettian blade, could demonstrate some similarities with "epi-aurignacian" characters [START_REF] Obadӑ | Preliminary results of studies of the Valea Morilor Upper Palaeolithic site (Chişinău, Republic of Moldova): A new camp of mammoth hunters[END_REF].

Material and methods

The studied material includes all the bones of large mammals from Valea Morilor preserved at the Institute of Zoology (Chişinău, Republic of Moldova). Several elements were altered during the search or during their removal. Part of the osteometric surveys were done in situ on long bones and tusks by T. Obadă. In order to be collected, the bones were consolidated with BF-6 and PVA glues. Several bones or group of bones were collected in block and cleaned in the laboratory of the Institute of Zoology. Other bones were discovered after sediments sieving.

A previous study was focused on the palaeontological data to identify the different species and define the population profile of woolly mammoths [START_REF] Demay | Large mammals from Upper Palaeolithic site of Valea Morilor (Republic of Moldova)[END_REF]. Osteometric measurements follow the procedures of von den [START_REF] Driesch | A guide to the Measurement of Animal Bones from Archaeological Sites[END_REF]concerning mammoth, of Agenbroad (1994), [START_REF] Lister | Sexual dimorphism in the mammoth pelvis: an aid to gender determination[END_REF] and Göhlish (1998). For the identification of mammoths (Mammuthus primigenius), age determination was based on epiphyseal stages of long bones and eruption and eruption/wear sequence of the jugal teeth [START_REF] Vaufrey | Proboscidiens fossiles[END_REF][START_REF] Coppens | Les élephants du Quaternaire français: dentition, systématique, signification et préhistoire[END_REF]Laws, 1996;[START_REF] Lister | Epiphyseal fusion and postcranial age determination in the woolly mammoth, Mammuthus primigenius[END_REF][START_REF] Haynes | Mammoths, Mastodonts and Elephants, Biology, behavior and the Fossil record[END_REF]. Five age classes have been defined: -juvenile: stage IXa ; from 0 to 8-12 years old -young adult: stages IXa-XVII ; from 12 to 18-26 years old -intermediate adult: stages XVII-XVIIIa ; 25-30 years old -mature adult: stages XVIIIa-XXVI ; 30-50 years old -old adult: stages XXVI-XXX death ; 50-60 years old The identification of sex is based on the morphometry of the bones. Concerning Elephantids, the males are generally more massive than the females [START_REF] Haynes | Mammoths, Mastodonts and Elephants, Biology, behavior and the Fossil record[END_REF][START_REF] Averianov | Sexual dimorphism in the mammoth skull, teeth, and long bones[END_REF]Shoshani, Tassy, 1996). For osteometric comparisons, we referred to Upper Pleistocene well-informed specimens from Eurasia (Russia, Germany and Britain).

Here we developed the zooarchaeological study, which also includes palaeontological analyzes, the biology and ethology of the extant species related to Pleistocene taxa. In addition, the description and quantitative analysis of the anatomical elements, associated with taphonomic studies (climato-edaphic and non-human biological factors) made it possible to identify the processes and circumstances in generating the deposit records. The combination of these analyzes, in relation to the possible human modifications, leads to a better understanding of the anthropogenic impact on this assemblage [START_REF] Poplin | Remarques théoriques et pratiques sur les unités utilisées dans les études d'ostéologie quantitative, particulièrement en archéologie préhistorique[END_REF][START_REF] Binford | Organization and Formation Processes: Looking at Curated Technologies[END_REF][START_REF] Behrensmeyer | Taphonomic and ecologic information from bone weathering[END_REF]1990;[START_REF] Patou-Mathis | Outillage peu élaboré en os et en bois de Cervidés IV[END_REF][START_REF] Lyman | Vertebrate taphonomy[END_REF][START_REF] O'connor | The Archaeology of Animal Bones[END_REF][START_REF] Reitz | Zooarchaeology[END_REF][START_REF] Denys | Manuel de Taphonomie[END_REF].

Concerning weathering we used stages defined by [START_REF] Behrensmeyer | Taphonomic and ecologic information from bone weathering[END_REF]: Taxonomic references and systematics are based on the zoological nomenclature code (1999).

Anatomical terms are used according to current nomenclature [START_REF] Barone | Anatomie comparée des mammifères domestiques : Ostéologie[END_REF]). Here we adopt the quantification units defined by [START_REF] Poplin | Remarques théoriques et pratiques sur les unités utilisées dans les études d'ostéologie quantitative, particulièrement en archéologie préhistorique[END_REF][START_REF] Lyman | Quantitative Paleozoology[END_REF]. The Minimal Number of Elements (MNE) is the representation of the skeletal elements for a taxon, not taking into account fragmentation phenomena. Complete elements are counted, taking into account refitting, pairing, age and sex. The skull (cranium and face) is considered as an element. The frontal appendices can also be considered as separate elements. The hemi-mandible is counted as an element, except for the mammoth whose mandible is considered as one element. A tooth, whether isolated or in place, is considered as an element.

The Minimal Number of Individuals (MNI) depends on the degree of determination of the bone assemblage. It is necessary to proceed to refitting, pairing, associations, according to the criteria of age and sex in order to maximize MNI.

The specific coefficient (Qsp) corresponds to the number of bone elements present in the complete skeleton of a species. The General Index of Skeletal Conservation permits to identify the skeleton preservation. It is the enumeration of the general conservation of the elements of a species, based on the ratio of presence of the cranial skeleton and post-cranial skeleton. This index can be obtained from the number of remains or the minimum number of elements. For comparison the theoretical indices are based on the specific coefficient of each element according to the species. Index = number of bones of cranial skeleton/number of bones of post-cranial skeleton (theoritecal index for woolly mammoth = 0,07) For the analysis of burned remains, we used different methods and experiment results. The characterization criteria defined by [START_REF] Costamagno | Combustible ou non ? Analyse multifactorielle et modèles explicatifs sur des ossements brûlés paléolithiques[END_REF] and [START_REF] Théry-Parisot | Un programme d'archéologie expérimentale en Taphonomie : la question des os brûlés dans les sites du Paléolithique[END_REF] take into account the percentage of burned bones less than 2 cm long, the percentage of burned cancellous bone and the percentage of at least carbonized bones. Categories of combustion are defined according to [START_REF] Stiner | Differential Burning, Recrystallization, and Fragmentation of Archaeological Bone[END_REF] and [START_REF] Clark | Burned bone in the Howieson's Poort and post-Howieson's Poort Middle Stone Age deposits at Sibudu (South Africa): behavioural and taphonomic implications[END_REF] (Table 2). The at least carbonized bones correspond to ≤ to code 2.

Results

The fauna of Valea Morilor is dominated by the woolly mammoth (90.3% of MNE), associated with at least a horse, a bison, a cervid, a bear and a polecat (Table 3).

Palaeological information

The woolly mammoth (Mammuthus primigenius) is the marker of a cold and open palaeoenvironment, generally associated with a plant cover composed of grasses, forbs and shrubs, such as sedge, sage, mugwort and dwarf birch [START_REF] Renault-Miskovsky | Le paysage végétal des mammouths durant les trois derniers millions d'années[END_REF].

The horse, whose species cannot be specified here, is not a good palaeoenvironmental marker. As for the bison, it can be adapted to very varied environments. The Bison priscus, now extinct, was adapted to a steppe environment and herbaceous food [START_REF] Guthrie | Frozen fauna of the Mammoth Steppe: the story of Blue Babe[END_REF][START_REF] Crégut-Bonnoure | Ordre des Artiodactyles. V. Famille des Bovidae[END_REF].

Four remains of a juvenile bear have been discovered in Valea Morilor. If it is a brown bear, this species lives in mixed forests and wooded steppes in temperate or moderately cold climates. However, it has the potential to adapt to different environments [START_REF] Couturier | L'ours brun, ursus arctos L. : Histoire naturelle, éthologie, chasse[END_REF][START_REF] Fosse | Taphonomie et paléoethologie des Ursidés pléistocènes[END_REF][START_REF] Jost | Le grand retour de l'ours[END_REF]. His diet is omnivorous and very varied. According to the osteometric comparisons of a tooth, it could belong to the species U. spelaeus (Demay and Obadă, 2018). The cave bear habitat was limited to mixed forests, sometimes characterized by reliefs, avoiding grassy plains and Mediterranean vegetation zones. This is why it is quite rare in the Pleniglacial cold and dry plains [START_REF] Arguant | Famille des Ursidae[END_REF].

At last, the polecat is found in the forest but especially in wet places bordering ponds and marshes [START_REF] Bouchardy | Le Putois[END_REF].

According to the faunal spectrum, Valea Morilor was situated in an open environment near a watercourse under a cold climate. The presence of bear in such environment may be questionable, especially if it is a cave bear.

Preservation of bones

The bones were covered by concretionned greyish and clay sands. In addition, the surface of the bones is altered (Figure 5A). Several mammoth bones were still anatomically connected, including an ulna and radius, thoracic and lumbar vertebrae, and phalanges (Figure 5B-C-D). In addition, both a right lunatum and a triquetrum belonging to a male fit together. They were found in the same square (A-11 of sector 1C). Mammoth bones and undetermined large-sized mammal(s) remains are characterized by transverse, longitudinal, and stepped fractures. Many bones have internal/external removals. Fracture edges are often blunt (Figure 6).

On the one hand, fissures were generated by sedimentary compression phenomena on the bones. Internal and external removals of long bone diaphyses may be due to anthropogenic fracturing, cryoturbation, trampling of large mammals, or subsurface bone distortion in soft sediments, which may be strongly linked to trampling. Fracture edges could be blunted by the action of water. The bones of medium-sized mammals are characterized by spiral fractures, generated on fresh or wet bones. The edges and surfaces are not blunted, it is unlikely that they are due to hydraulic transport. However they may have been generated by trampling, or cryoturbation. Finally, they may be fractures due to carnivore chewing or human breakage.

According to the study of the modifications (Figure 7; Table 4) due to climato-edaphic agents, the bones are highly altered by weathering (stage 1-2), at a higher degree for the mammoth remains (stages 4-5) . Indeed, the surfaces are desquamated, exfoliated and cracked. Surfaces are heavily impregnated with iron and mostly manganese oxides. Many bones show alteration due to dissolution, related to runoff water and mineral and organic acids. All the bones whith more preserved surfaces exhibit traces of rootlets. Bones with more altered surfaces may also have been affected by plants. There is little evidence of charriage-à-sec or trampling. Only one remain, a fragment of mammoth ulna, was chewed by a small carnivore. Pronounced and localized orange colorations were identified on the bones. In addition, orange-colored indurated elements have been identified within the sediments.

It may be imported coloring matter used by humans. However, they are small and associated with black spots and the dissolution indices are high. It is rather residual deposits due to the differential alteration of other elements. These characteristics are typical of hydromorphic soils, subject to a regular saturation in slow water, which has physico-chemical consequences on the environment. This saturation in water may be due to a slug of water or to the variation of the bed of a river. In periglacial context, permafrost is also subject to the water change of state, with alternating seasons. The site of Valea Morilor is located in the discontinuous permafrost area [START_REF] Vandenberghe | The Last Permafrost Maximum (LPM) map of the Northern Hemisphere: permafrost extent and mean annual air temperatures, 25-17 ka BP[END_REF][START_REF] Schaetzl | Approaches and challenges to the study of loess -Introduction to the Loess-Fest Special Issue[END_REF]. In addition, the presence of clay sediments makes the soil more impervious, increasing the stagnation of water. The movements of the water, associated with the type of sediments, in particular the sands, with the presence of a plant cover and bacteria, increase pH and thus favor the concentration of ferro-manganic ions [START_REF] Ardouin | Guide pratique pour la description des sols de France[END_REF][START_REF] Berthier | Diagnostic in situ de la réduction du fer dans les sols par l'utilisation d'un test de terrain colorimétrique[END_REF]. The site of Valea Morilor is located in a basin, near a watercourse. Together with the type of sediments, it constitutes a highly favorable environment for this process.

According to the size classes of identified species and undeterminate splinters by sectors (258 remains), classes I (26%) and II (51%) (those smaller than 5 cm) are generally the most represented. We have few remains from sector 1A. Sector 1B is dominated by classes I (42%) and II (41%), then III (8%), IV (7%) and V (2%). Finally, sector 1C has mainly class II (61%), then IV (13%), I (12%), III (8%) and V (7%) classes.

The smallest size classes are generally associated with a human (fracturation, combustion) or carnivore intervention. The other classes could be related to climato-edaphic phenomena. The different agents must be responsible for the changes that have occurred within this assemblage.

Skeletal preservation and structure of animal populations

A horse is represented by six dental elements, belonging to at least a juvenile of less than 3 years old.

A bison is represented by two elements, a fragment of bone and a left M3 belonging to a mature adult, who was between 6 and 15 years old.

A bear is represented by four dental elements. The dimensions correspond rather to those of a cave bear (Demay and Obadă, 2018). According to the eruption and wear stages, it is a juvenile of about 1 year.

A polecat is represented by a left mandible fragment, with M1.

A phalanx of a small mammal, possibly belonging to a large mustelid (G. gulo or M. meles) and a fragment of antler were also identified.

Woolly mammoth (M. primigenius) is represented by 588 remains, from at least 206 elements (Table 5). According to refitting and possible pairing, mammoths are represented by at least six individuals. Almost all the anatomical elements are represented, except the sacrum and the caudal vertebrae. It should also be noted that, with the exception of tusks, no skull remains were found. In addition, the bones are not very fractured. The most fragmented bones are tusks, cheek teeth and ribs, which are naturally more fragile, due to their morphology and structure.

According to the skeletal conservation indices, the elements are generally not very well preserved. The mandibles, the atlases, and the long bones of the anterior and posterior limbs are the best preserved (Figure 8). The general index of skeletal preservation shows that according to the number of remains, the cranial remains are quite strongly represented, whereas according to the elements the trend is similar to a natural preservation: IGCSNISP = 38/202= 0,2 IGCSNME = 13/90= 0,09 According to the age determination, mammoths are only adults: two young adults, one adult sensu lato, one adult female, and two mature adult males.

Human modifications

Butchering activities on elephantids are difficult to identify. Indeed, the mass of meat and fat of these animals restricts the effects of butchering activities on the bones, which leave few or ever no cutmarks [START_REF] Scott | Two Hunting Episodes of Middle Palaeolithic Age at La Cotte de Saint-Brelade, Jersey (Channel Islands)[END_REF][START_REF] Crader | Recent single-carcass bone scatters and the problem of « butchery » sites in the archaeological record[END_REF]. Moreover, the surfaces of the bones of Valea Morilor are altered.

A fractured rib of mammoth is broken in two pieces. The depression on the proximal part was interpreted as a groove due to human shaping to insert lithic pieces (Obadă et al., 2011;[START_REF] Ardouin | Guide pratique pour la description des sols de France[END_REF]), in Maszycka Cave, Poland (Kozlowski et al., 2012). Here we think that it could be a pathology or/with taphonomic alteration processes. This rib was discovered in sector 1C, several meters from a hearth. It might have been sawn, however the fracture edge is damaged and covered with indurated sediments, leaving no possibility of reliable interpretation (Figure 9A). An undetermined bone fragment bears cutmarks. Its surface is blunt, due to hydraulic phenomena. It is likely to be in a secondary position (Figure 9B). There are 249 burned bones (16.7% of the assemblage). Most of them are attributed to large-sized mammals, including woolly mammoths. Bone is the most burned material, with the use of mammoth ribs. Seven fragments of cheek teeth, from two mammoth and one horse and four mammoth ivory fragments were also burned (Figure 10). Apart from dental fragments, burned bones are mostly smaller than 5 cm (classes I and II), then longer than 10 cm (class IV). Class IV remains are long bones especially ribs of large-sized mammal, including mammoths, which have been partially burned. Burned bones are mostly carbonized (98%). They are associated with few and moderately burned bones, as well as moderately calcined (Figure 11). Burned bones smaller than 2 cm are present in sectors 1B and 1C, as well as remains with an unspecified sector (Table 6). The sector A is very poor so not representative. According to the types of bone tissue, burned remains composed of cortical and spongiosa bone are the best represented (Table 7).

Discussion

Preservation of the assemblage

According to the taphonomic characters, the Valea Morilor bones were buried rather quickly in subsurface, but more slowly for those of mammoths. In addition, a plant cover was present. The bones are impregnated with iron and manganese oxides by post-depositional hydromorphy and percolation effects. They have also been subject to the effects of hydraulic alteration phenomena related to the variations of the river bed. Fractures may have been due to cryoturbation. Concerning faunal remains all the anatomical parts are represented, little dispersed, sometimes still connected. So, according to the palaeoenvironmental context highlighted by the geomorphological, pedological and malacological studies and our taphonomic observations, the archaeological and faunal remains were in a floodplain characterized by weak current.

Origin of the accumulation of mammoth bones

According to the study of bone preservation, all the anatomical elements are present. This observation associated with the fact that there are several elements still in anatomical connection, allow to affirm that the mammoths died in situ. There is no selection of bones, it is a natural preservation. The absence of cranial elements raises questions. Cranium is subject to fragmentation and splinters are easily dispersed in case of hydraulic movements, which is not the case for the maxillary and especially the upper cheek teeth, which could also due to human transportation.

The number of individuals is too small to show a significant mortality profile. However, the assemblage contemporaneity raises questions. In fact, mature males are rather solitary, with young adult males being in a group. In addition, the presence of a mature female does not correspond with the known social behaviors of extant elephants, which live in separate groups between males and females during their maturity. It could be a one-time grouping, however it is unlikely to have all three types of social groups on the same site. The accumulation likely results from temporally close events rather than strictly ones.

It is quite possible that the bone remains found at Valea Morilor are natural accumulations of dead animals near a water point.

Extant elephantids are gregarious animals living in herds, consisting of adult females, young adults, and juvenile males and females. Around the age of 12, young males leave the group. Occasionally, older males are reintegrated into the group [START_REF] Pfeffer | Vie et mort d'un géant : L'éléphant d'Afrique. Flammarion, collection[END_REF]. Young adult males are the most vulnerable individuals to predators according to data from current elephant groups [START_REF] Haynes | Mammoths, Mastodonts and Elephants, Biology, behavior and the Fossil record[END_REF]. Indeed, at this time, they have little ability to find sources of water and food during the early days. Moreover, males are slower-growing so they often have to find more resources. In addition, they suffer a lot of stress due to the lack of knowledge of their new environment.They may associate themselves with older males wich have a good knowledge of the territories. By the way, during musth periods their metabolism is upset and their aggressivity greatly increases. They are exhausted and confront other individuals, which can lead to serious injuries (fractures, infections) and can develop many autoimmune diseases (anemia, loss of the immune system) [START_REF] Poole | Musth in the African elephant, Loxodonta Africana[END_REF][START_REF] Poole | Announcing intent: the aggressive state of musth in African elephants[END_REF]. Too much stress can also lead to their death. They can also become trapped in muddy areas or holes, without being able to get help usually brought in the matriarchal herds. After a few years when life expectancy is reduced, males become larger, they know their environment better. They are therefore avoided by predators and better manage resources access. However, the musth period is lengthening and the hormonal changes are increased, thus worsening the sanitary conditions described above. Males are therefore more likely to experience natural mortality.

The matriarchal herds, led by an experienced female, are characterized by a strong sense of mutual aid. The calves, which represent in addition an important biological investment, are highly protected. Even if it can happen, it is less common that an adult female is attacked by predators, injured or isolated in natural traps.

Mammoth at Valea Morilor might also have been slaughtered by palaeolithic hunters. The lack of core would mean that they came with their weapons. In addition, the splinters of retouches tend to show that lithic elements were reworked in situ. The presence of a fractured scraper raises questions about its potential use on site.

Woolly mammoths were between 2.50 and 3.50 m at the withers, weighed between 3 and 4 tons, and had large spiral tusks that ranged between 2.50 m and 3.50 m long. They were very massive animals, had a very great physical strength, especially mature individuals. In addition they had thick fur, a thick skin (3 cm thick) and a layer of fat (8 cm thick). Finally, the solidarity that characterizes elephantids is a very important criterion, especially when a matriarchal group is hunted. These characteristics should make any approach dangerous and be a major obstacle to the effectiveness of hunting projectiles. As with the other elephantids, the skeleton of the woolly mammoth is characterized by the vertical stacking of the legs, which gives them this particular structure called in column, to support the general mass of the animal. The massive body is therefore well supported, but the ability to rotate at the top of the leg is weak [START_REF] Hildebrand | Energy of the oscillating legs of a fast-moving cheetah, pronghorn, jackrabbit, and elephant[END_REF], which negates the ability to race and jump [START_REF] Shoshani | Elephants: the super keystone species[END_REF]. It could, probably like the current elephants, move fast (up to 40 km / h) and perform loads against predators. It is notable that elephants avoid climbing hills or crossing mountainous areas. Indeed, climbing slopes represents a considerable energy cost for these animals. Moreover, as they face their predators, it is then possible to shoot them by alternating shots, being several people.

Evidence of elephantid hunting is not always easy to put forward. However, it is demonstrated or suspected in several sites around the world, practiced by different hominids, from ethological, biological and ethnographic references [START_REF] Agam | Elephant and Mammoth Hunting during the Palaeolithic: A Review of the Relevant Archaeological, Ethnographic and Ethno-Historical Records[END_REF]. It may be a direct association between a projectile and bones, or age profiles indicative of human intervention [START_REF] Haynes | Mammoths, Mastodonts and Elephants, Biology, behavior and the Fossil record[END_REF][START_REF] Haynes | Finding meaning in mammoth age profiles[END_REF], also by the techno-typology of associated lithic artefacts [START_REF] Gaudzinski | The Use of Proboscidean Remains in everyday Palaeolithic Life[END_REF]. The site of Lehringen (Germany) bears witness to the practices of the Neanderthals' hunting of the straight-tusked elephant, notably the presence of a yew point thrown in a carcass [START_REF] Thieme | Neue untersuchungen zum eemzeitlichen Elefanten-Jagdplatz Lehringen[END_REF]. Upper Palaeolithic hunting by H. sapiens is strongly considered, particularly the Yudinovo (Russia) [START_REF] Germonpré | Possible evidence of mammoth hunting during the Epigravettian at Yudinovo, Russian Plain[END_REF], Milovice [START_REF] Péan | Comportements de subsistance au Gravettien en Europe centrale : (Autriche, République tchèque, Pologne, Hongrie)[END_REF][START_REF] Brugère | L'exploitation du mammouth à Milovice et dans le Gravettien de Moravie (République Tchèque)[END_REF]), Krakow Spadzista (Kufel-Diakowska et al., 2016) and Pushkari 1 (Demay et al., 2015) sites. The Lugovskoe and Yana (Russia) sites provided the most obvious evidence for the discovery of woolly mammoth remains in which lithic points are embedded [START_REF] Maschenko | The Lugovskoe site: relations between the mammoth assemblage and Late Palaeolithic man[END_REF][START_REF] Maschenko | La Question de la chasse au mammouth : une preuve de l'abattage des mammouths dans un site du Paléolithique supérieur au nord de la Sibérie occidentale[END_REF][START_REF] Nikolskiy | Evidence from the Yana Palaeolithic site, Arctic Siberia, yields clues to the riddle of mammoth hunting[END_REF].

In Valea Morilor, a smooth, rounded perforation on the lateral surface of an ulna was found, belonging to a mature adult male or female (Demay and Obadă, 2018). It measures 14.5 x 8.6 mm and is 27 mm deep (Figure 20). It was filled with sediment. Another alteration was caused by a modern metal tool. Perforations of this dimension on bones can be due to carnivores, impacts of hunting projectiles or can be a cloaca due to a pathology. It is too narrow to have been provoked by the defense of another individual.

According to the shape and depth of the perforation, the absence of other marks (incisors, cracks, spirals) (Fernández-Jalvo and Andrews, 2016), the hypothesis of the intervention of a carnivore can be dismissed.

The hypothesis of an impact caused by the tip of a hunting projectile was advanced (Obadă et al., 2012). (It is here the opinion of T. Obadă.) Published experiments have been conducted to highlight the damage caused by hunting weapons on bones. These may include hand-thrown projectiles (javelins) or thrusters (spears), or spears used as a thrusting weapon. In general, the damage to the bones of juveniles differs from that of adults in relation to the degree of mineralization of the bone; the juvenile bones have a greater tendency to burst [START_REF] Letourneux | Hunting lesions caused by osseous projectile points: experimental results and archaeological implications[END_REF]. Shot experimentations were made with Solutrean projectile points (shoulder points, antler points) on medium-sized herbivore carcasses [START_REF] Morel | Impacts de chasse et archéozoologie. Quelques observations expérimentales[END_REF] and large herbivores (cow and horse) [START_REF] Castel | Identification des impacts de projectiles sur le squelette des grands ongulés[END_REF]. The stigmas left by the flint tips are scraping, bursting / dislocation, cracking / splitting, implantation / insertion and perforation. Marginal scrapes and splits are much more common on the bones of large-sized mammals than on those of medium-sized mammals [START_REF] Castel | Identification des impacts de projectiles sur le squelette des grands ongulés[END_REF]. The antler points [START_REF] Morel | Impacts de projectile sur le gibier: quelques éléments d'une approche expérimentale[END_REF]Smith et al., 2007, Letourneux and[START_REF] Letourneux | Hunting lesions caused by osseous projectile points: experimental results and archaeological implications[END_REF] can cause scratches, piercings, incrustations and fissures.

Wood could be used directly, especially for javelins and spears. These are the most powerful weapons in terms of impact damage [START_REF] Cnuts | Extracting residues from stone tools for optical analysis: towards an experiment-based protocol[END_REF][START_REF] Coppe | Focus on the target. The importance of a transparent fracture terminology for understanding projectile points and projecting modes[END_REF]. It usually takes hard wood. Both techniques cause similar damage, including step fractures on ribs and vertebrae, spiraling on the long bones, and piercing on scapulas [START_REF] Smith | Damage inflicted on animal bone by wooden projectiles: Experimental results and archaeological implications[END_REF].

There is no cracking or fracturing on the Valea Morilor specimen and the edges of the perforation are smooth. In addition, the cortical and the spongiosa thickness are important in the proximal part.

These is due to pathological conditions, particularly an infectious lesion that could have damaged the bone, in the form of a cloaca. These disorders are known in modern elephant populations [START_REF] Fowler | The Biology, Medicine and Surgery of Elephants[END_REF], as well as fossil assemblages of woolly mammoths including Milovice and Kraków Spadzista [START_REF] Krzemińska | Abnormalities and Other Changes on Woolly Mammoth Bones from Central Europe 32,000 to 20,000 Years Ago: a Monograph[END_REF] and early proboscidians (de Souza [START_REF] De Souza Barbosa | Osteological diseases in an extinct Notiomastodon (Mammalia, Proboscidea) population from the Late Pleistocene of Brazil[END_REF].

Use of mammoth resources

One or more carcasses could have been scavenged by humans in Valea Morilor. Recovery of bone marrow is discussed. In fact, in woolly mammoth, the long bone medullary canal is narrow and dense in bone, with few or without marrow, leaving only a thin passage where haematopoiesis, the manufacture of blood cells, takes place. This allowed the bones to withstand greater body pressure than other mammals and to keep relative mobility. There would have been no interest for human groups to break long bones. However, marrow is found in the trabecular architecture of the bone, which could be an important contribution for human groups [START_REF] Fladerer | Hearth-side bone assemblages within the 27 ka BP Krems-Wachtberg settlement: Fired ribs and the mammoth bone-grease hypothesis[END_REF].

The origin of burned bone remains can be multiple. This may be wildfire [START_REF] Clark | Burned bone in the Howieson's Poort and post-Howieson's Poort Middle Stone Age deposits at Sibudu (South Africa): behavioural and taphonomic implications[END_REF], cooking remains [START_REF] Speth | Hunting and overhunting in the Levantine late middle Palaeolithic[END_REF], use as fuel [START_REF] Costamagno | Taphonomic consequences of the use of bones as fuel. Experimental data and archaeological applications[END_REF], Théry-Parisot et al., 2005), wastes thrown into the fire or bones that were near or under the hearth [START_REF] Stiner | Differential Burning, Recrystallization, and Fragmentation of Archaeological Bone[END_REF][START_REF] Cain | Using burned animal bone to look at Middle Stone Age occupation and behavior[END_REF][START_REF] Clark | Burned bone in the Howieson's Poort and post-Howieson's Poort Middle Stone Age deposits at Sibudu (South Africa): behavioural and taphonomic implications[END_REF][START_REF] Bosch | Humans, bones and fire: Zooarchaeological, taphonomic, and spatial analyses of a Gravettian mammoth bone accumulation at Grub-Kranawetberg (Austria)[END_REF].

Moreover some bones could have been prepared for the within-bone fats and after be put in the hearth [START_REF] Fladerer | Hearth-side bone assemblages within the 27 ka BP Krems-Wachtberg settlement: Fired ribs and the mammoth bone-grease hypothesis[END_REF]. According to experiments [START_REF] Costamagno | Taphonomic consequences of the use of bones as fuel. Experimental data and archaeological applications[END_REF]2009;[START_REF] Théry-Parisot | The use of bone as fuel during the Palaeolithic, experimental study of bone combustible properties[END_REF], whole bones burn better. Also, the complete epiphyses are more effective than the fragments, because the fat is released more quickly than in the fragmented epiphyses. According to other experiments [START_REF] Yravedra | Burnt bone assemblages from El Esquilleu cave (Cantabria, Northern Spain): deliberate use for fuel or systematic disposal of organic waste?[END_REF], the elements of the axial skeleton (vertebrae, ribs and girdles) are also good fuels. The preponderance of spongiosa elements corresponds more particularly to the use of bones as fuel [START_REF] Théry-Parisot | The use of bone as fuel during the Palaeolithic, experimental study of bone combustible properties[END_REF]. The equal representation of spongiosa bone and cortical bone would be consistent with throwing bones as waste to clean the camp [START_REF] Théry-Parisot | Un programme d'archéologie expérimentale en Taphonomie : la question des os brûlés dans les sites du Paléolithique[END_REF]. In Valea Morilor, both types of tissue are equally represented, including rib fragments.

In Valea Morilor the site was occupied with low intensity use and management. So, the presence of burned bones is probably not due to cleaning process of the camp. Finally, the bones are small-sized fragments mainly carbonized. So it does not correspond to cooking remains. Some bones were partially burned, so they were probably located near hearth and another part was used as fuel.

In Valea Morilor, it is certain that mammoth bones were used as fuel by humans. There may have been a consumption of soft matter combined with the use of bones as a combustion material. It can also be gathered bones. In this case human would have come with food resources. The bone remains of other species associated with this assemblage may come from natural and / or human processes.

The use of dry bone as a combustible material is controversial. Dry bone can be used if the fire has already reached relatively high energy [START_REF] Glazewski | Experiments in Bone Burning[END_REF], but does not participate in combustion [START_REF] Costamagno | Taphonomic consequences of the use of bones as fuel. Experimental data and archaeological applications[END_REF][START_REF] Yravedra | Burnt bone assemblages from El Esquilleu cave (Cantabria, Northern Spain): deliberate use for fuel or systematic disposal of organic waste?[END_REF]. The presence of grease will cause combustion, which is why fresh bone can be used as a combustible material. Mammoth bones, including ribs, have been used as combustible material in several sites, including Kraków Spadzista, Poland (Wojtal et al. (eds.), 2015), Mezhyrich, Ukraine) [START_REF] Péan | Mammouth et comportements de subsistance à l'Épigravettien : analyse archéozoologique du secteur de la fosse no 7 associée à l'habitation no 1 de Mezhyrich (Ukraine)[END_REF] and Krems-Wachtberg, Austria [START_REF] Fladerer | Hearth-side bone assemblages within the 27 ka BP Krems-Wachtberg settlement: Fired ribs and the mammoth bone-grease hypothesis[END_REF]. It is the same during the contemporary occupations of the Upper Pleniglacial of the East European Plain [START_REF] Demay | New evidences about human activities during the first part of the Upper Pleniglacial in Ukraine from zooarchaeological studies[END_REF].

However the degree of bone "freshness" can not be specified; it can be very fresh bones, or bone gathered several months or a few years after the death of the animal. In Valea Morilor fresh bones could have been used and then mammoth carcasses remains in quite long open air conditions. Or it could be dried carcasses from which bones were gathered and use for fire.

Human settlements

The Palaeolithic site of Valea Morilor probably corresponds to a short-term occupations, perhaps several occupations. The presence of a bear raises questions. Regarding bears, calving takes place in the den in winter season. According to the age of the individual found at Valea Morilor, his death occurred during the same period the following year. If it was killed by humans, it means they occupied this location in winter.

Regional settings

Contemporary human settlements of Valea Morilor mainly furnished gravettian material. The Gravettian of the Dniester valley is characterized by the appearance of shouldered points, the development of burins on retouched truncation and microgravettes. There are no aurignacian tools such as nosed end-scrapers, carenated end-scrapers, aurignacian blades, nor Mousterian artifacts such as scrapers, Mousterian points and denticulates. There are few bifacial pieces, Kostienki knives, leaf-shaped points and pavlovian shouldered points. Furthermore shouldered pieces are narrower than those Kostienki type (which are large, with elongated shoulder and reverse retouch). This type of shouldered points and the composition of the lithic material permits to identify an original regional culture called Molodovian or eastern Gravettian. There are typological changes during the Epigravettian, but the laminar production techniques are similar to those of the Gravettian (Boriskovsky, 1953;Grigor'ev, 1970;[START_REF] Chernysh | Paleolit i mezolit Pridnestrov'ya (karty i katalog mestonakhozhdeniy)[END_REF][START_REF] Otte | Le Gravettien en Europe Centrale, 2 volumes, Dissertationes Archaeologicae Gandenses[END_REF][START_REF] Otte | Recherches sur le Paléolithique supérieur de la Moldavie[END_REF]Borziac and Koulakovska, 1998;[START_REF] Borziac | Considérations concernant le Gravettien de l'espace compris entre le Dniestr et les Carpates[END_REF][START_REF] Djindjian | Ruptures et continuités dans les industries du maximum glaciaire en Europe centrale et orientale: la question de l'Epigravettien[END_REF]Noiret, 2004;2007a;[START_REF] Haesaerts | The east Carpathian loess record: a reference for Middle and Late Pleniglacial stratigraphy in Central Europe[END_REF]Nuzhnyi, 2009).

In the Dniester area between 30 000-14 000 BP reindeer is the most commonly exploited species. However, several sites from Ukraine, Romania and Republic of Moldova are characterized by:

-the presence of mammoth bones: Molodova V, Babin I, Korman IV, Cosăuţi, Raşkov 7 and 8, Voronovitsa I, Mitoc-Malu Galben and Mejigirzy [START_REF] Chernysh | Pozdnii paleolit Srednego Pridnestrov'ya[END_REF][START_REF] Chernysh | Mnogosloynaya paleoliticheskaya stoyanka Molodova-5[END_REF][START_REF] Tatarinov | Vertebrate fauna of Korman IV site[END_REF]David, in Borziac 1991;1993;Gautier and López Bayón, 1993;David, Obadă and López Bayón, in Borziac, 1996;Bibikova and Belan, in Kulakovska and Otte, 1998;Borziac and Obadă, 1999;[START_REF] David | Late Pleistocene fauna from the Late Palaeolithic butchering site Cosӑuți I[END_REF][START_REF] Noiret | Le Paléolithique supérieur de la Moldavie. Essai de synthèse d'une évolution multi-culturelle[END_REF][START_REF] Croitor | Mammal fauna from Upper Palaeolithic site of Raşcov-8 (Moldova). Oltenia. Studiile şi comunicării[END_REF]; -dominating the faunal spectrum: Climăuţi II and Galich [START_REF] David | Restes squelettiques de mammifères dans les fouilles de la station paléolithique de Climӑuți[END_REF][START_REF] Wojtal | The new Upper Palaeolithic mammoth site at Halich (Ukraine)[END_REF] -ivory use to make tools and artistic pieces: Dorochivtsy III [START_REF] Koulakovska | Dorochivtsy III-Gravettian site in the Dniester valley (Ukraine)[END_REF]Demay et al., 2015).

The lithic assemblage of Valea Morilor is quite poor. If it appears to be a particular facies with epi-aurignacian characters, this phenomenon has been observed in several sites (Raşkov VII, Climăuți II), without any direct cultural affiliation between them. The Epi-Aurignacian corresponds to the resurgence of techniques used in the Aurignacian, in particular by the presence of burins and carinated microliths. For some authors, it seems difficult to give a similar techno-typological name to these various complexes (Covalenco, 2003(Covalenco, -2004;;[START_REF] Zwyns | La problématique de l'Aurignacien tardif dans la zone des steppes nord-pontiques / The late Aurignacian problematic in the north Pontic steppe zone[END_REF]Noiret, 2007b;Chirica and Valeanu, 2007). Moreover in Valea Morilor the occupation(s) are not oriented to reindeer exploitation in relation with migration. So for the moment it remains difficult to determine the cultural relations between Valea Morilor and the other regional occupations.

So in order to have a better understanding of Valea Morilor site, we need to make new datations of charcoals. Furthermore we need to process to new studies about faunal remains from Climauti II to bring new informations about human settlements, exploitation of fauna and particularly mammoth status.

Conclusions

The site of Valea Morilor is a new spot concerning upper Palaeolithic human occupations in the Dniester valley. According to zooarchaeological analyses, the fauna is dominated by the woolly mammoth, associated with a horse, a bison, a cervid, a bear and a polecat. Mammoths are represented by at least six individuals died in situ: two young adults, one adult sensu lato, one adult female, and two adult males, which correspond to different mortality events in a flood plain in periglacial context. While we can neither confirm nor deny the use of the mammoth as a food resource, we identified use of bones, whose mammoth bones, as combustible during at least a halt, perhaps several halts. This site would correspond to nomad road within a circulation area. Bones and here particularly mammoth bones are a good resources to establish camps.

Figure 1 .

 1 Figure 1. Localisation of the palaeolithic site of Valea Morilor in Republic of Moldova and view on excavations (Obadӑ et al., 2012).

Figure 2 .

 2 Figure 2. Stratigraphy of the eastern wall of Valea Morilor pit n°4 (Obadӑ et al., 2011). 1: heterogeneous chernozem with krotovina. (40 cm) 2: brown loam with carbonate inclusions and krotovina. The transition with the lower unit is progressive. (50-70 cm) 3: uniform yellowish-brown loam, of alluvial origin. Krotovina are present in the upper part. (around 1.6 m) 4: clay loams (in grey) alternating with river sands (in white). (40 cm)

Figure 3 .

 3 Figure 3. Flint tools of Valea Morilor. (Covalenco in Obadӑ et al., 2011) 1: scraper; 2: retouched blade; 3: burin

Figure 4 .

 4 Figure 4. Planigraphic records of the remains from the three sectors of Valea Morilor excavation n°1. 1: mammoth bones and from other species; 2: concentrations of ashes and charcoals (after Obadӑ, Covalenco and Burlacu, 2011). Ashy area with charcoals from Valea Morilor sector 1B, interpreted as a hearth (Obadă et al., 2012).

Figure 5 .

 5 Figure 5. Preservation of bones from Valea Morilor. A: concretionned mammoth rib; B: mammoth thoracic vertebrae; C: mammoth lumbar vertebrae; D: mammoth phalanges.

Figure 6 .

 6 Figure 6. Distal end and metaphysis of mammoth femur from Valea Morilor, in posterior view, with details on the internal removal and the blunt edge, in anterior views.

Figure 7 .

 7 Figure 7. Alterations due to climato-edaphic and non-human biological agents in percentage of the total number of remains from Valea Morilor and focus on the weathering.

Figure 8 .

 8 Figure 8. Skeletal preservation by anatomical parts in percentage of minimum animal unit (%MAU) and percentage survival (Ps%) of mammoth from Valea Morilor.

Figure 9 .

 9 Figure 9. Bones from Valea Morilor. A: mammoth rib; B: bone fragment with cutmarks.

Figure 10 .

 10 Figure 10. Representation in percentage of burned bones by species and categories and type of bones from Valea Morilor.

Figure 11 .

 11 Figure 11. Burned bones in number of remains by size classes and combustion degrees from Valea Morilor.

Figure 12 .

 12 Figure 12. Perforation on the lateral face of a mammoth left ulna from Valea Morilor and x-ray picture.

Table 1 . C-14 dating from palaeolithic site of Valea Morilor.

 1

	sector	dating 14 C BP	reference	material
	1A	20 770 ± 90 GrA-46004	mammoth bone
	1B	20 570 ± 80 GrA-52424	mammoth bone
	1C	20 560 ± 80 GrA-52425	mammoth bone

Table 2 . Burning damage categories based on macroscopic appearance and colour (after Stiner et al., 1995; Clark and Ligouis, 2010).

 2

	Burn colour code	Description
	0	not burned (cream/tan)
	1	slightly burned (<50% black)
	2	lightly burned (>50% black)
	3	fully carbonized (100% black)
	4	localized (<50% white)
	5	half calcined (>50% white)
	6	fully calcined (100% white or blue-grey)

Table 3 . Counting of faunal remains from Valea Morilor in number of identified specimens (NISP), minimal number of element (MNE) and minimal number of individual (MNI).

 3

	Species	NISP MNE	MNI
	M. primigenius	588	206	6
	Equus sp.	6	6	1
	Bison sp.	2	2	1
	Bovine	2	2
	Cervid	1	1	1
	Ursus sp. (cf. spelaeus)	4	4	1
	M. putorius	2	2	1
	identifiable	605	225
	Large-sized mammal	497
	Large-or medium-sized mammal	169	1
	Medium-sized mammal	50
	Small-sized mammal	9	3	1
	Herbivores	28
	Carnivores	2	1
	Undetermined splinters	130
	TOTAL	1490	228	12

Table 4 . Alterations due to climato-edaphic and non-human biological agents in number by species from Valea Morilor and focus on the weathering stages.

 4

	species	weathering	percolating	runoff water /	charriage-à-sec/	plant roots	carnivore

Table 5 . Counting of mammoth remains from Valea Morilor.

 5

	Elements	Qsp NISP		MNE
			L R Und. Tot.
	tusk	2	20		4	4
	mandible	1	4		4	4
	lower cheek teeth	6	4 2	2		4
	undetermined cheek teeth	12	10		1	1
	teeth	14	14			0
	cranial skeleton	18	38 2	2	9	13
	atlas	1	5		5	5
	axis	1	2		2	2
	cervical vertebra	5	2		2	2
	undetermined vertebra	52	27		19	19
	thoracic vertebra	19	19		16	16
	lumbar vertebra	4	8		8	8
	rib	38	56 7	1	24	32
	axial skeleton	91	119 7	1	76	84
	scapula	2	9 1	2	1	4
	humerus	2	6 3	1	2	6
	radius	2	2 2			2
	ulna	2	9 2	1	3	6
	anterior upper part	8	26 8	4	6	18
	lunatum	2	3 1	2		3
	triquetrum	2	3	3		3
	pisiform	2	2	2		2
	trapezum	2	1	1		1
	trapezoid	2	1	1		1
	hamatum	2	2	2		2
	carpals	16	12 1 11		12
		10	5 2	3		5
	III	2	3 2	1		3
	IV	2	2	2		2
	ant. basipod and metapod	26	5 2	3		5
	carpals/metacarpals	26				0
	pelvis	2	3 1		1	2
	femur	2	8 3	4		7
	patella	2	2		2	2
	tibia	2	4	2	2	4
	posterior upper part	10	17 4	6	5	15
	talus	2	4 2	1	1	4
	navicular	2	6 3	3		6
	cuboid	2	3 1	1	1	3
	internal cuneiform	2	1 1			1
	meso-cuneiform	2	2	2		2
	tarsals	14	16 7	7	2	16
	metatarsals	10	2	1	1	2
	I	2	1	1		1
	II	2	1		1	1
	post. basipod and metapod	24	18 7	8	3	18
	tarsals/metatarsals	24				0
	carpals/tarsals	30	5		5	5
	undetermined metapodial	20	7		7	7
	undetermined phalanges	56	1		1	1
	prox.	20	8		8	8
	int.	16	5		5	5
	dist.	20	2		2	2
	sesamoid	40	1		1	1
	acropod et sesamoids	96	17		17	17
	post-cranial skeleton	255	202 28 22	90	140
	Qsp: specific coefficient; NISP: number of identified specimens ; MNE: minimum number of elements; L: left;
	R: right; Und. : undertermined; Tot.: total.

Table 6 . Percentage of burned bones smaller than 2 cm from Valea Morilor.

 6

		1A	1B	1C	undetermined sector	total
	number of remains	3	8	13	47	71 (29%)

Table 7 . Types of bone tissue represented among burned remains from Valea Morilor.

 7

		1A	1B	1C	undetermined sector	Total
	1: cortical bone	0	12	2	0	14 (5,8%)
	2: spongiosa bone	0	0	2	1	3 (1,2%)
	3: cortical and spongiosa bone	3	15	27	180	225 (92,9%)

Acknowledgement

We thank I. Toderaş, as well as all members of the Institute of Zoology of the Academy of Sciences of Moldova in Chişinӑu, for their availability and hospitality. This study was conducted as part of an ATM project (Thematic Actions of the Museum) 2016, led by S. Péan, entitled Use of animal resources by the the last Neanderthals and the first Homo sapiens in Moldavia and Western Ukraine. We also thank S. Covalenco, V. Burlacu, J. Van der Plicht, M. Patou-Mathis, P. Haesaerts, P. Noiret, H. Salomon and J. Coppe (University of Liège), for their advice. We thank la Société des Amis du Musée de l'Homme for the Leroy Gourhan Prize. We also thank the organizors of the VIIth International Conference on Mammoths and their Relatives, Taichung (Taïwan), 17-23 septembre 2017, who permitted us to present a part of our results.


Sophie Alleaume
email: sophie.alleaume@aphp.fr

Catherine Tardy

François Richard

Dochead dossier Sous-dochead Le temps au bloc opératoire Surtitre enquête L'optimisation des blocs opératoires, une priorité de l'AP-HP

Keywords: audit, concordance de temps, enquête, optimisation, organisation, performance

Fort enjeu pour l'Assistance publique-Hôpitaux de Paris, la qualité des soins et la performance des blocs opératoires ont bénéficié d'un dispositif d'optimisation en 2016. Une enquête, réalisée sur un jour en 2017, avait pour objectifs de comparer ses résultats à ceux de 2015. Un recentrage de l'heure d'arrivée des professionnels cohérente avec celle du patient a été constaté, de même qu'une amélioration des temps d'enchaînement. Cette enquête a permis également d'identifier des points perfectibles.

La qualité des soins et la performance des blocs opératoires représentent un enjeu fort pour l'Assistance publique-Hôpitaux de Paris (AP-HP). Consciente de cet enjeu, l'organisation, qui comprend 21 hôpitaux chirurgicaux, 50 blocs opératoires et 287 salles, s'est engagée dans une démarche volontariste définissant l'optimisation des blocs opératoires comme l'un des 16 projets prioritaires de son plan stratégique. Début 2016, le dispositif d'optimisation des blocs opératoires a été renforcé à l'occasion de la mise en place d'une mission sur les évolutions de la chirurgie dans les hôpitaux de l'AP-HP.

T1 Contexte

Le bloc opératoire, haut lieu de technicité et plateforme à haut risque, est aussi un lieu d'interfaces complexes qui nécessite une organisation et une coordination garantissant la qualité et la sécurité des soins. Les enjeux organisationnels et de management des relations humaines sont forts. Le bloc opératoire constitue une zone de collaboration et parfois de confrontation de compétences humaines spécialisées, cependant toutes légitimes (chirurgicales, anesthésiques, soignantes, logisticiennes…).

T1 Objectifs

Une enquête relative à la concordance des temps entre les différents professionnels contribuant à la prise en charge du patient a été réalisée en 2015 et renouvelée en 2017 au sein des blocs opératoires de l'AP-HP. Les objectifs étaient de mesurer l'évolution des organisations par rapport à la précédente enquête, d'analyser le déroulement d'une journée et de vérifier la qualité de mise en oeuvre de la check-list pour les phases 1 et 2 la concernant.

T1 L'enquête

TEG1 Sur un jour donné, l'enquête ciblait les salles d'opérations de chirurgie programmée, hors prise en charge des urgences, transplantations et endoscopies.

TEG1 Concernant la phase 1 de l'enquête, relative aux démarrages tardifs, des constats encourageants, notamment sur la concordance des temps montrent un recentrage de l'heure d'arrivée des professionnels cohérente avec celle du patient (figure 1). Quant aux délais d'installation du patient et de l'heure d'incision, leur amélioration a contribué à l'optimisation des prises en charge.

Si une amélioration significative a été constatée, il reste toutefois 25 % des salles (contre 38 % en 2015) qui ont démarré avec 16 à 60 minutes de retard par rapport à l'heure initialement prévue. Parmi, les causes recherchées de ce retard, la principale était liée aux personnels. TEG1 La phase 2 de l'enquête, centrée sur le déroulement de la journée et les temps peropératoires, a constaté une amélioration des temps d'enchaînement des phases opératoires par rapport à l'enquête précédente (figure 2). Cependant trois points restent perfectibles liés au début du geste opératoire, à la durée du bionettoyage et au délai entre les interventions.

T1 Résultats

Les résultats de l'enquête ont été adressés et présentés à l'ensemble des parties prenantes de l'organisation des blocs opératoires des hôpitaux de l'AP-HP.

Des échanges formalisés avec les acteurs des blocs opératoires ont permis la mise en place d'actions prioritaires pour :

• renforcer le respect des modalités de réalisation de la check-list ;

• mettre en place des formations obligatoires à destination des cadres de bloc, des directeurs et des présidents des conseils de blocs ;

• organiser la formation récurrente sur l'utilisation du logiciel du bloc opératoire ;

• amélioration de la concordance temps ;

• perfectionner la programmation opératoire ;

• consolider les bonnes pratiques et réaliser des audits.

Dans le cadre de son plan stratégique, l'AP-HP poursuivra sa dynamique d'amélioration de l'organisation des blocs opératoires en réitérant chaque année cette enquête un jour donné auprès des équipes.

Déclaration de liens d'intérêts

Les auteurs déclarent ne pas avoir de liens d'intérêts.

Figures All_fig1 Figure 1 .Figure 2 .

 All_fig112 Figures


Stéphanie Perez
email: stefany031@hotmail.fr

Dochead matériovigilance/gestion des risques Surtitre sécurité Analyse de gestion des risques dans le cadre des prélèvements d'anatomopathologie

Keywords: fixation des prélèvements, formation, gestion des risques, Ibode, ischémie, sensibilisation

Dans le cadre de leur formation Ibode, un groupe d'élèves de Toulouse a réalisé une affiche de sensibilisation aux bonnes pratiques quant à la gestion des prélèvements en anatomo-pathologie, où l'Ibode joue un rôle primordial. Le travail s'est concentré, pour l'exemple, sur les pièces anatomiques dans le cadre du traitement d'une forme particulière de cancer du sein.

De plus, cela s'inscrit dans les objectifs du Plan national pour la sécurité des patients 2013/2017 [2] qui promeut notamment l'implication des professionnels de santé et l'initiation de méthodes dans la prévention des risques.

T1 Constat de départ TEG1 Les questions au sujet du traitement des prélèvements d'anatomo-pathologie sont quotidiennes au bloc opératoire :

• Quel fixateur utiliser ?

• Quel contenant pour le prélèvement ?

• Quel délai pour acheminer le prélèvement au laboratoire ?

Toutes ces questions aboutissent à une problématique : quelles sont les bonnes pratiques en ce qui concerne les prélèvements d'anatomo-pathologie au bloc opératoire ? TEG1 À partir de ce constat, il est apparu nécessaire de créer un support afin de fournir ces différentes informations directement aux professionnels qui en ont besoin.

En effet, les pratiques inadéquates observées sur le traitement de ces prélèvements ne sont pas faites par malveillance ou manque de compétence, mais bien par ignorance à la fois des bonnes pratiques mais aussi des enjeux pour le patient. À ce jour, des affiches existent, cependant elles sont peu nombreuses et conçues par des médecins anatomo-pathologistes à la demande des laboratoires.

La plus-value de ce projet réside dans le fait qu'il est pensé par des infirmiers pour des infirmiers.

T1 Démarche du projet

TEG1 L'idée de départ a été de créer une affiche contenant toutes les informations sur le traitement des prélèvements d'anatomo-pathologie mais également compatible avec le bloc opératoire.

TEG1 Cette affiche se base sur les recommandations de 2014 du Groupe d'étude des facteurs pronostiques immuno-histochimiques dans le cancer du sein (GEFPICS) [START_REF] Penault-Llorca | Mise à jour 2014 des recommandations du GEFPICS pour l'évaluation du statut HER2 dans les cancers du sein en France2014 update of the GEFPICS' recommendations for HER2 status determination in breast cancers in France[END_REF]. Il s'agit de médecins anatomo-pathologistes qui émettent des recommandations techniques en qualité d'expert.

TEG1 Une fois la forme validée, les informations importantes sur les bonnes pratiques ont été recherchées et listées par les élèves de la promotion 2014-2016 de l'école d'infirmiers de bloc opératoire de Toulouse. Elles ont ensuite été validées par des médecins anatomo-pathologistes de la région Midi-Pyrénées.

TEG1 Le circuit du prélèvement

TEG1 Il est intéressant d'expliquer ces bonnes pratiques au travers d'un exemple, afin de prendre pleinement conscience que les actions que nous faisons au quotidien dans nos blocs opératoires sont décisives pour l'analyse ultérieure des prélèvements.

TEG1 Le parcours d'un prélèvement se divise en trois phases majeures (figure 1) :

• Pré-analytique, qui se déroule en partie au bloc opératoire, c'est elle qui a été retenue pour le projet des élèves Ibode. Elle comprend la prise en charge immédiate du prélèvement, sa fixation ou non, son conditionnement et sa transmission. Elle s'étend donc de l'exérèse à l'arrivée de la pièce au laboratoire. Le rôle de l'Ibode est primordial durant cette phase puisqu'il en est responsable.

• D'analyse des prélèvements et de diagnostic, celle-ci a lieu au laboratoire d'anatomopathologie et dépend des techniciens, du cadre, de l'ingénieur et des pathologistes.

• Rédaction du compte rendu par les pathologistes.

L'issue de ce parcours mène aux choix thérapeutiques.

T2 L'exemple de HER2

TEG1 Afin d'illustrer notre propos, l'exemple du prélèvement dans le cadre du cancer du sein a été retenu, plus précisément le type HER2 de cette maladie. Nous connaissons bien les cancers du sein hormonosensibles à la progestérone ou aux oestrogènes, cependant 15 % des patientes atteintes d'un cancer du sein surexpriment 2 une protéine. Celle-ci est un récepteur transmembranaire appelé HER2 (figure 2) 3 . 4 du centre Jean Perrin à Clermont-Ferrand précise qu'en « cas d'emballement de HER2, on constate une agressivité plus importante de la cellule tumorale, des rechutes plus rapides de la maladie, la survenue plus fréquente de métastases et une résistance de la tumeur aux thérapeutiques conventionnelles ». Cependant ces cancers bien particuliers peuvent bénéficier de thérapie ciblée.

TEG1 Frédérique Penault-Llorca

TEG1 Pour adapter le traitement aux femmes qui en ont besoin, il est donc capital de savoir si les tumeurs présentent trop de récepteurs HER2 (il est alors question de statut HER2 positif). L'objectif premier du pathologiste dans ce cas consiste à mettre en évidence la potentielle surexpression de cette protéine. Pour cela, il est parfois nécessaire d'analyser directement l'acide désoxyribonucléique (ADN), il faut alors un prélèvement de très bonne qualité. En effet, l'analyse génomique se développe dans beaucoup de spécialités et si, aujourd'hui, les pathologistes s'intéressent à l'ADN, demain ce sera à l'acide ribonucléique (ARN).

TEG1 Ce matériel est fragile et sa dégradation dépend de plusieurs facteurs. Une sous-fixation du prélèvement peut, par exemple, induire de faux-négatifs, c'est-à-dire que des patientes surexprimant HER2 et qui devraient avoir une thérapie ciblée sont diagnostiquées négatives.

Le pathologiste n'a pas le moyen de savoir qu'il est en face d'un faux négatif. La sous-fixation induit également des faux négatifs dans le dépistage des cancers hormonosensibles. Là encore, le traitement qui suit ne sera pas approprié, ce qui impacte à la baisse l'espérance de vie de ces patientes.

TEG1 Le problème de la durée de fixation illustre bien l'enjeu pour les patientes. Bien entendu, ce n'est pas le seul élément qui importe dans l'analyse du prélèvement aussi, tous les facteurs requis pour un prélèvement de qualité sont présents sur l'affiche du projet. TEG1 Dans le cas de prélèvement fixé, il importe de savoir qu'une sous-fixation est irréversible. Le temps de fixation optimal étant compris entre 6 et 72 heures. Les techniciens et pathologistes n'ont qu'une seule source d'information afin de connaître le temps de fixation : le bon de transmission que l'infirmier de bloc opératoire rempli, notamment l'heure de fixation. TEG1 C'est pourquoi, l'affiche comprend un tableau en trois parties (figure 3), afin de bien comprendre la conduite à tenir concernant :

T1

• la pièce opératoire et son conditionnement ;

• la traçabilité du prélèvement sur le bon de transmission ;

• l'attitude de l'Ibode dans ce cadre.

TEG1 Au bas de ce tableau, chaque établissement a la possibilité de noter le contact du laboratoire d'anatomo-pathologie. En effet, ce qui ressort de ce projet, c'est qu'en dehors des recommandations il existe des pratiques dans certains blocs opératoires qui sont validées par les chirurgiens et les pathologistes. Comme le fait de fixer systématiquement le prélèvement en salle d'intervention, par exemple. L'essentiel réside dans la communication entre le laboratoire et le bloc opératoire, entre le prélèvement et l'analyse. TEG1 À gauche de ces informations, les temps de traitements des prélèvements sont symbolisés par un feu tricolore. TEG1 Afin d'obtenir une bonne lisibilité, le format A3 a tout de suite été retenu ainsi que l'orientation paysage. De plus, le texte a été réduit au maximum grâce à l'utilisation d'images. La plastification rend le nettoyage facile et permet l'affichage en salle d'intervention en respectant les règles d'hygiène au bloc opératoire.

T1 Conclusion

TEG1 La conception de cette affiche et sa présentation aux professionnels de bloc opératoire a pour but de faire connaître les bonnes pratiques, mais surtout de sensibiliser les Ibode aux enjeux pour le patient. C'est pourquoi, il était important de concrétiser ce projet. Grâce à la création d'une association par les élèves, des fonds ont été levés et plus de 100 affiches imprimées. Elles ont été distribuées dans différents blocs opératoires mais également lors d'événements comme les Journées régionales d'études et de perfectionnement en 2015 à Toulouse et la journée de formation "Actualisation des connaissances" dispensée par le Centre hospitalier universitaire de Toulouse dans le cadre du développement professionnel continu. TEG1 À la suite des interventions des élèves lors de ces journées, une centaine de professionnels, infirmiers, Ibode et cadre de santé Ibode ont été sensibilisés. Depuis, certains établissements de santé de l'ensemble du territoire ont spontanément réclamé l'affiche et les retours sont positifs. Afin d'assurer la continuité de cette action la promotion 2015/2017 de notre école d'infirmiers de bloc opératoire a repris le projet afin de continuer à le présenter lors d'événements professionnels et de poursuivre la diffusion de l'affiche dans les blocs opératoires.

  Les enjeux TEG1 Les enjeux dépendent du temps d'ischémie froide, à savoir la période entre la résection opératoire et le placement dans un fixateur et ce, que la mise dans le fixateur se passe en salle d'intervention sur demande du chirurgien ou au laboratoire d'anatomo-pathologie. En effet, à terme toutes les pièces seront fixées au laboratoire. Durant cette période d'ischémie froide, le prélèvement subit dessiccation et dégradation. C'est pourquoi, il faut soit fixer rapidement la pièce anatomique au bloc opératoire. Si c'est une pièce dite fraîche, elle doit partir au laboratoire dans les plus brefs délais.

Figures Per_fig1 Figure 1 .Figure 2 .Figure 3 .

 Per_fig1123 Figures Per_fig1 Figure 1. Parcours d'un prélèvement dans lequel l'Ibode joue un rôle primordial durant la phase préanalytique. © S. Perez/Elsevier Masson SAS Sur 2 colonnes près de son appel

000 récepteurs HER2 présents à la surface de la cellule

Expression physiologique de HER2 qui conduit à une croissance et à une prolifération régulées des cellules

Jusqu'à 2 millions de récepteurs HER2 présents à la surface de la cellule Surexpression de HER2 qui entraîne une croissance et une prolifération excessives des cellules

Cellule saine

Cellule cancéreuse HER2+