Trace element (Cd, Cu, Hg, Se, Zn) accumulation and tissue distribution in loggerhead turtles (Caretta caretta) from the Western Mediterranean Sea (southern Italy)

Keywords: Bioaccumulation, Trophic transfer, Heavy metals, Marine vertebrates, Sea turtles, Tyrrhenian Sea

Cadmium (Cd), copper (Cu), mercury (Hg), selenium (Se) and zinc (Zn) were determined in the liver, kidney and muscle of 29 loggerhead turtles, Caretta caretta, from the South Tyrrhenian Sea (Western Mediterranean). Cd and Hg exhibited a wider range of concentration in the examined tissues than Cu, Se and Zn. For all the elements analysed, no significant differences (p > 0.05) were detected between males and females. Trace element concentrations were not influenced by the size of the specimen except Se in the liver, which was negatively correlated with the curved carapace length (p < 0.001). The distribution of the analysed elements was comparable to that reported in other marine vertebrates. Thus, the muscles generally displayed the lowest trace element burdens, with the exception of Zn reaching elevated concentrations as high as 176 µg g -1 dry wt. Kidneys displayed the highest Cd and Se mean concentrations (57.2 ± 34.6 and 15.5 ± 9.1 µg g -1 dry weight, respectively), while liver exhibited the highest Cu and Hg values (37.3 ± 8.7 and 1.1 ± 1.7 µg g -1 dry weight, respectively). Whatever the tissue considered, toxic elements had elevated coefficients of variation (i.e. from 60% to 177%) compared to the essential ones (i.e. from 14% to 65%), which is a consequence of homeostatic processes for Cu, Se and Zn. Globally, the concentrations of Hg remained low in all the considered tissues, which might be the result of low trophic level in sea turtles. In contrast, the diet of loggerhead turtles would result in a significant exposure to Cd.

Highly significant correlations between Cd and Cu and Zn in the liver and kidney suggest that efficient detoxification processes involving MTs occur to prevent Cd toxicity in loggerhead turtles.

Introduction

Trace elements are natural components of rocks and soil and enter the marine environment as a consequence of weathering and erosion [START_REF] Garrett | Natural source of metals of the environment[END_REF]. Many of them are biologically essential but all have the potential to be toxic to biota above a threshold concentration [START_REF] O'shea | Toxicology in marine mammals[END_REF]. Following industrialization, unnatural quantities of such elements have been released, and continue to be released into the sea altering the natural biological equilibrium [START_REF] Haynes | Organochlorine, heavy metal and polyaromatic hydrocarbon pollutant concentrations in the Great Barrier Reef (Australia): a review[END_REF].

Therefore some concern has arisen regarding their possible adverse effects on marine wildlife, particularly on those long living species such as marine mammals, seabirds and sea turtles, which have the potential to accumulate these contaminants [START_REF] Caurant | Bioaccumulation of Cadmium, Copper, and Zinc in some tissue of three species of marine turtles stranded along the French Atlantic coasts[END_REF]Anan et al., 2001). Although the first two taxa have been thoroughly studied, only a limited number of papers on trace element accumulation in sea turtles have been published to date (see the review of Storelli and Marcotrigliano, 2003).

Because of their solitary life style, duration of the pelagic phase and long lasting apnoeas, sea turtles are in fact among the most difficult marine animals to assess (Witherington, 2003).

Two of the seven species of sea turtles are known to reproduce regularly in the Mediterranean Sea, the loggerhead turtle, Caretta caretta, and the green turtle, Chelonia mydas [START_REF] Groombridge | Marine turtles in the Mediterranean: distribution, population status, conservation[END_REF]. The former is the most abundant and extends its distribution to the whole basin with Western Mediterranean waters providing some of the most important feeding grounds (Margalitoulis et al., 2003). The neritic juvenile stage and adult foraging stage occur in the neritic zone [START_REF] Bolten | Active swimmers-passive drifters: The Oceanic juvenile stage of loggerheads in the Atlantic system[END_REF].

Turtles feed primarily on sessile or slowly moving benthic preys [START_REF] Mortimer | Feeding ecology of sea turtles[END_REF]Bentivegna et al., 2001) although they do capture organisms throughout the water column [START_REF] Bolten | Active swimmers-passive drifters: The Oceanic juvenile stage of loggerheads in the Atlantic system[END_REF].

The loggerhead turtle is currently classified as "vulnerable" by IUCN (International Union for the Conservation of Nature and Natural Resources), but it has been suggested to be considered as "endangered" in the Mediterranean Sea [START_REF] Broderick | Population and nesting ecology of the green turtle, Chelonia mydas, and the loggerhead turtle, Caretta caretta, in northern Cyprus[END_REF].

Because of the slow renewal of its waters, the large concentrations of human populations along its coasts, the intense maritime activities and the presence of natural sources of pollutants (Bacci, 1989), levels of contaminants in the Mediterranean sea are considered to be relatively high [START_REF] Turley | The changing Mediterranean Sea -a sensitive ecosystem?[END_REF]. For example several studies on different taxa revealed an enhancement of Hg in cephalopods, fish or dolphins compared to other areas [START_REF] Renzoni | Mercury concentration in the water, sediments and fauna of an area of the Tyrrhenian coast[END_REF]Bernhard, 1988;André et al., 1991). However the paucity of data regarding trace element burdens in loggerhead turtles from the Mediterranean Sea (Godley et al., 1999, Storelli et al., 1998ab) makes difficult the evaluation of accumulation levels and the comparison among different sites.

The primary objective of this study was therefore to determine the concentrations of five trace elements, Cd, Cu, Hg, Se, and Zn, in the tissues of loggerhead turtles from the Western Mediterranean Sea and to compare these data with those reported from other locations. Secondly, the influence of sex and size on trace element concentrations was tested. Correlations between the elements were determined in order to investigate physiological disorders and the hazard that these pollutants pose to loggerhead turtles survival were discussed.

Materials and methods

Sampling and sample preparation

Tissue samples were taken during necropsies of 29 loggerhead turtles, which stranded dead along the South Tyrrhenian coasts (Western Mediterranean) between March 2000 and May 2001 (Fig. 1). The curved carapace length (CCL), measured to the nearest 1 cm using a flexible meter tape, ranged from 37 to 82 cm. The necropsies were undertaken within 24 hours after the detection of the carcass. Liver, kidney, and pectoral muscle were sampled when possible in a minimum quantity of 20 g and stored in plastic bags to prevent from any contamination. The samples were frozen at -20 °C until the chemical analysis. The sex was determined via visual examination of the gonads. The CCL, sex, sampled tissue and the sampling date of each turtle are shown in Table 1. Tissue samples were freeze dried at -60 °C and 6 mbar. Fresh and dry weights were determined and the water content, expressed as a percentage of fresh weight was calculated.

Analytical procedures

Approx. 200-300 mg of each homogenized dry sample was heated with supra-pure nitric acid at 60 °C until the solution was clear. Then, the residues were completed to 10 ml with ultrapure Milli-Q quality water. All the elements except Hg were determined by Flame or Graphite Furnace Atomic Absorption Spectrophotometry (AAS) with a Varian spectrophotometer Spectra 250 Plus. Hg analysis was carried out with an Advanced Mercury Analyser spectrophotometer, ALTEC AMA 254, which does not require an acid-digestion of the samples.

Aliquots ranging from 10 to 50 mg of dried samples were directly analysed after they had been inserted into the oven of the apparatus. After drying, the samples were heated under oxygen atmosphere for 3 minutes and Hg was subsequently amalgamated on a gold-net. Then the net was heated after a 45 sec. waiting time to liberate the collected Hg, which was measured by AAS.

Standards of dogfish liver DOLT-2 of the NRCC (National Research Council of Canada) were analysed as analytical quality control using the same procedure as the samples. Recoveries of all elements ranged from 93% to 110%. Trace element concentrations are presented as µg g -1 of tissue on dry weight (dwt) basis.

Data analyses

Because the data were not normally distributed, statistical analyses of the data were performed by non parametric tests with Minitab 11.2 (Minitab Inc., Pennsylvania). The coefficients of variation (CV) were calculated. The Wilcoxon's test for matched pairs was used to detect differences in the accumulation of trace elements between sex and the Kruskall-Wallis test was used to evaluate the organotropism of each element. Spearman rank coefficients were calculated between pairs of elements in each tissue and between trace element concentrations and CCL.

A p value of less than 0.05 was considered to indicate statistical significance [START_REF] Fowler | Practical statistics for field biology[END_REF].

Results

Tissue concentrations

Cd, Cu, Hg, Se and Zn concentrations in the tissues of loggerhead turtles from the Tyrrhenian Sea are reported in Table 2. As no significant differences in trace element concentrations have been found between sexes and size (with the exception of hepatic Se which was negatively correlated with the CCL, Table 3), results in Table 2 were presented together. Trace element concentrations were generally low in muscle, except for Zn which exhibited the highest mean concentration in this tissue.

Kidney displayed the highest Cd and Se concentrations, reaching up to 158 and 41.8 µg g -1 dwt, respectively. Finally, the liver exhibited the highest Cu and Hg loads, reaching up 41.8 and 8.76 µg g -1 dwt, respectively. Toxic elements (i.e. Cd and Hg) were separated from those essential (i.e. Cu, Se, and Zn) according to their coefficients of variation of the concentrations (CV). Indeed, CV range from 60 to 177% for Cd and Hg while they remained lower than 65% for Cu, Se and Zn (Table 2). The mean water content was 68%, 76%, 79% for liver, kidney and muscle, respectively.

Trace element correlations

Table 3 shows the correlations between trace elements within the three tissues. In kidney, Zn was correlated with Cd (p < 0.001), Hg (p < 0.001) and Se (p < 0.05) (Table 3). Renal Hg concentrations were also positively correlated to Cu (p < 0.05) and to Cd (p < 0.05) (Table 3). In liver, Cd concentrations were correlated to Hg (p < 0.05) and Zn (p < 0.001), (Table 3). Only hepatic Se was correlated with the CCL (p < 0.001) (Table 3).

Discussion

Sea turtles can achieve life span greater than 50 years, and have a potential to bioaccumulate heavy metals and pesticides [START_REF] Lutcavage | Human impacts on sea turtles survival[END_REF]. In the costal zone, chronic pollution from industrial, agricultural wastes, and urban runoff constitutes a threat to sea turtles (National Research Council, 1990). Unfortunately, there is little information about this problem. The present results showed that although the Mediterranean Sea is subjected to relative high inputs of pollutants [START_REF] Kuetting | Mediterranean pollution[END_REF][START_REF] Turley | The changing Mediterranean Sea -a sensitive ecosystem?[END_REF], trace element concentrations in specimens from the Tyrrhenian Sea (South Western Mediterranean) are comparable or even lower than those determined in the same species from other areas (Table 4). For comparative purposes, data which were originally presented on a wet weight basis were converted to approximate dry weights using the mean water content of each tissue determined in this study (see Results section). In general Cu, Se, Zn concentrations in the tissues of loggerhead turtles did not vary much among sites (Table 4). Consistently, the CV of these elements were the lowest found in all the analysed tissues (Table 1).

Therefore it appears that loggerhead turtles control Cu, Se, and Zn concentrations through homeostatic processes in a balance between metabolic requirements and prevention against toxic effects. Consequently, human activity is not likely to be a factor in the accumulation of these elements. On the contrary geographical area influences Cd and Hg concentrations especially in those organs like liver and kidney, which are known to play a main role in their long term accumulation (Table 4). The high CV found (Table 2) imply that Cd and Hg levels are not actively controlled by turtles and should change according to the level of exposure. In this context it is noteworthy that Hg concentrations in all specimens analysed remained very low for an area supposed to have natural mercury enrichment [START_REF] Renzoni | Mercury concentration in the water, sediments and fauna of an area of the Tyrrhenian coast[END_REF]Bacci, 1989;André et al., 1991;[START_REF] Capelli | Study of trace elements in organs and tissues of striped dolphins (Stenella coeruleoalba) found dead along the Ligurian coasts (Italy)[END_REF]. More generally, loggerhead turtles do not accumulate Hg to such a great extent (Table 4) as other long living marine vertebrates [START_REF] Caurant | Ecological and biological factors controlling the concentrations of trace-elements (As, Cd, Cn, Hg, Se, Zn) in delphinids Globicephala melas from the North-Atlantic Ocean[END_REF]Cardellicchio et al, 2002). Since this metal is known to biomagnify in the marine food web the absence of long term Hg accumulation suggests that this species, which is an opportunistic predator [START_REF] Tomas | Marine debris ingestion in loggerhead sea turtles, Caretta caretta, from Western Mediterranean[END_REF], feeds constantly on preys of low trophic level. This is confirmed by investigations of the stomach and intestine contents of loggerhead turtles in the study area, which revealed that their diet is composed mainly of molluscs and crustaceans (Bentivegna et al., 2001). Such diet involves only few bioaccumulative steps and generally displays low organo-Hg contents compared to a fish based diet [START_REF] Cappon | Chemical form and distribution of mercury and selenium in edible seafood[END_REF].

Cd mean concentrations were comparable within the Mediterranean Sea but lower than those found in other ocean locations (Table 4). However this element which contrary to mercury does not biomagnify [START_REF] Gray | Biomagnification in marine systems: the perspective of an ecologist[END_REF], accumulates at levels comparable to those reported for other long living marine vertebrates [START_REF] Caurant | Ecological and biological factors controlling the concentrations of trace-elements (As, Cd, Cn, Hg, Se, Zn) in delphinids Globicephala melas from the North-Atlantic Ocean[END_REF]. In marine mammals or sea birds, such concentrations (and also higher) are often encountered in animals feeding mainly on squid which are considered to be an important vector of this element to top marine predator [START_REF] Bustamante | Cephalopods as a vector for the transfer of cadmium to top marine predators in the north-east Atlantic Ocean[END_REF].

Also loggerhead turtles are most likely to take up Cd via their diet but the paucity of data about the total Cd content and its physico-chemical form within the prey tissues prevents from any final statement.

Because no reliable age determination method exists for sea turtle (Bjorndal et al., 1998), we used the curved carapace length (CCL) to evaluate growth related variations in trace element concentrations. Although we analysed the so far largest sample of loggerhead turtles, we did not detect any clear growth related trends in trace element accumulation, the only exception being Se (Table 3). This was surprising, since elements such as Cd and Hg are expected to accumulate with age because of their long biological half life [START_REF] Gray | Biomagnification in marine systems: the perspective of an ecologist[END_REF]. However such trends were previously reported in both green (Chelonia mydas) and hawksbill turtles (Eretmochelys imbricata) [START_REF] Gordon | Trace metal concentrations in livers and kidneys of sea turtles from south-eastern Queensland, Australia[END_REF]Sakai, 2000b;Anan et al., 2001).

Consequently we believe that the size effect in our sample was concealed by the unequal distribution of specimens throughout the size classes, and by the absence of young and very old turtles (CCL < 35 cm and CCL > 82 cm). The early juvenile loggerhead turtles foraging in open ocean pelagic habitats (Bjorndal, 1997) may in fact experience different trace element exposure than older animals which feed on benthic preys.

The results of this study show that sex has no significant influence on trace element concentrations in loggerhead turtles. Although no other study has analysed such dependence in this species, no gender differences in trace element concentrations were also observed by Anan et al. (2001) in both green and hawksbill turtles. It might be probable that sexual differences in feeding rates, metabolism or growth rates are reduced in these species.

Overall, no particularly high concentrations of trace element in loggerhead turtles have been found during this study. This included Hg, one of the most dangerous marine pollutants, which has been found at very low levels compared to those reported in other long living marine organisms. Although loggerhead turtles show high hepatic Cu concentrations, several authors agreed that these levels are physiologically controlled (Sakai et al., 2000a;Storelli and Marcotrigliano, 2003) as further suggested by the low CV found in the present study (Table 2). Therefore Cu is not likely to affect the health of these endangered animals. The only exception may be the very toxic Cd, which accumulates at relatively high concentrations compared to those reported for other marine vertebrates [START_REF] Nicholson | Kidney lesions in pelagic seabirds with high tissue levels of cadmium and mercury[END_REF][START_REF] Caurant | Cadmium contamination in pilot whales Globicephala melas: source and potential hazard to the species[END_REF]Elinder and Järup, 1996;Storelli and Marcotrigliano, 2003). However the significant relationship between Cd and Zn in both liver and kidney (Table 3) suggests the involvement of metallothionein proteins (MTs) in the prevention of Cd toxic effects (WHO, 1992 ;[START_REF] Vogiatzis | Cadmium accumulation in liver and[END_REF]ASTDR, 1999a). The presence of these molecules has been recently verified in loggerhead turtle (Anan et al., 2002). Further studies are necessary to evaluate physiological effects of Cd on loggerhead turtles and the potential tolerance to this toxic element via MTs induction because disturbance due to a chronic exposure are expected to happen at a lower concentrations than the toxic limit.

Conclusion

The loggerhead turtles from the Tyrrhenian Sea displayed similar concentrations of trace elements to those reported from other areas. Surprisingly, the Hg concentrations in their tissues remained very low even in the biggest ones (up to 82 cm of length) for an area supposed to be largely contaminated. This result confirms that the loggerhead turtles feed constantly on low trophic level preys, as revealed by stomach content analysis. However, these prey probably accounted for a significant Cd exposure, which is accumulated in relatively high amounts in the kidney and liver. Highly significant correlations between Cd and Cu and Zn in the liver and kidney suggest that efficient detoxification processes involving MTs occur to prevent Cd toxicity in loggerhead turtles.

Acknowledgements. This study was supported by the Stazione Zoologica Anton Dohrn and the Laboratoire de Biologie et Environnement Marins. We gratefully acknowledge the field support provided by Gianfranco Mazza, Mariapia Ciampa and Angela Paglialonga We thank Sandra Hochscheid for her constructive comments on the manuscript.

Reference list

Anan, Y., Kunito, T., Watanabe, I., Sakai, H., Tanabe, S., 2001. Trace element accumulation in hawsbill turtles (Eretmochelys imbricata) and green turtles (Chelonia mydas) from Yaeyama islands, Japan. Environ. Toxicol. Chem. 20, 2802-2814. Anan, Y, Kunito T., Sakai H, Tanabe S., 2002. Subcellular distribution of trace elements in the liver of sea turtles. Mar. Pollut. Bull. 45, 224-229. Andrè, J., Boudou, A., Ribeyre F., Bernhard, M., 1991. Comparative study of mercury accumulation in dolphins (Stenella coeruleoalba) from French Atlantic and Mediterranean coasts. Sci. Tot. Environ. 104, 191-209. ASTDR., 1999a. Toxicological Bjorndal, K., Bolten, A., Bennet, R., Jacobson, E.R., Wronski, T.J., Valeski, J.J., and Eliazar, P.J., 1998. Age and growth in sea turtles: limitations of skeletochronology for demographic studies. Copeia 1, 23-30. Bentivegna, F., Ciampa, M., Mazza, G., Paglialonga, A., Travaglini, A., 2001 Elements ------Liver -----------Kidney -----------Muscle -----

  Profile For Cadmium. Prepared for U.S Department of Health and Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry (ASTDR). Bacci, E., 1989. Mercury in the Mediterranean. Mar. Pollut. Bull. 20, 59-63. Bernhard, M., 1988. Mercury in the Mediterranean. UNEP Reg. Seas Rep. Studies, No. 98. Bjorndal, K.A., 1997. Foraging ecology and nutrition of sea turtles. In: Lutz, P.L., Musick, J.A. (Eds), The Biology of Sea Turtles.CRC press,

Figure legends: 1 Fig. 1 :

 11 Figure legends: 1

.

  Loggerhead turtle (Caretta caretta) in Tyrrhenian Sea: Trophic role of the Gulf of Naples. Proceedings of the 1st Mediterranean Conference on Marine Turtles, Rome, Italy.kidneys and hepatic metallothionein and glutathione levels in Rana ridibunda, after exposure to CdCl 2 . Arch.Environ. Contam. Toxicol. 34,[64][65][66][67][68]

	Witherington, B.E., 2003. Biological conservation of loggerheads: challenges and
	opportunities. In: Bolten, A.B., Witherington, B.E. (Eds), Loggerhead Sea Turtles.
	Smithsonian Institution, USA, pp. 295-312.
	WHO, 1992. Cadmium -Environmental Aspects. Vol. 135. Geneva, Switzerland:
	World Health Organization, International Programme on Chemical Safety.

Table 1 .

 1 Size (curved carapace length), sex and tissue sampled of 29 loggerhead turtles found dead 1 on the South Tyrrhenian coast of Italy between 2000 and 2001. (K=kidney, L=liver, M=muscle, 2

	3	ND=not determined)
		Specimen	Sex	Size (cm)	Tissue	Date of sampling
		1	♀	75	K, L, M	24-05-00
		2	♂	45	K, L, M	26-05-00
		3	ND	ND	M	05-06-00
		4	ND	71	K, L, M	11-06-00
		5	♀	58	K, L, M	12-06-00
		6	♂	54	L, M	18-06-00
		7	♀	44	K	23-06-00
		8	♂	43	L, M	27-07-00
		9	ND	63	L, M	07-08-00
		10	♀	71	L, M	"
		11	♂	64	K, L, M	10-08-00
		12	♂	57	K, M	11-08-00
		13	♂	62	K, L, M	16-08-00
		14	♀	62	K, L	21-08-00
		15	♀	71	M	26-08-00
		16	♂	53	L, M	01-09-00
		17	ND	ND	K, L,M	16-09-00
		18	♀	67	K, L, M	22-09-00
		19	♀	58	L, M	03-10-00
		20	♂	ND	K, L, M	04-10-00
		21	♀	37	K, L, M	20-10-00
		22	ND	ND	M	03-11-00
		23	♂	59	K, L, M	20-11-00
		24	♀	70	K, L	"
		25	♀	77	K, L, M	29-11-00
		26	ND	ND	K, M	03-01-01
		27	♂	82	K, L, M	04-01-01
		28	♀	51	K, M	16-01-01
		29	♀	73	K, L, M	19-03-01

Table 2 .

 2 Trace element concentrations (µg g -1 dwt) in the tissues of loggerhead turtles from the South Tyrrhenean coast of Italy. , n: number of samples, CV: coefficient of variation (%).

Table 3 .

 3 Spearman correlations between trace elements within the tissues of the loggerhead turtlesCaretta caretta from the South Tyrrhenian coast of Italy. Not underlined: p < 0.05 ; underlined: p < 0.001. CCL: curve carapace length.

	Elements	Liver	Kidney	Muscle
	Cd	+Zn, +Hg	+Zn, +Hg	+ Se
	Cu		+ Hg
	Hg	+Cd,	+Cd, +Cu, +Zn,
	Se	-CCL	+ Zn	+ Cd
	Zn	+Cd,	+Cd, +Hg, +Se

Table 4 .

 4 Reported trace element concentrations (µg g -1 dwt) in the tissues of loggerhead turtles from different locations. n : number of samples. 1Values reported in wet weight were converted to dry weight using the mean water content as determined in present study 2 3

		Cd		Cu	Hg	Se	Zn
	Location	n Mean ± SD	Range	n Mean ± SD Range	n Mean ± SD Range	n Mean ± SD Range	n Mean ± SD	Range	Reference
	Liver
	Australia	51.2 ± 10.3	22.8-110.0	-

0.05 ± 0.02 0.0-0.10


Elise De

Ph.D La Rochebrochard

M.Sc Céline Quelen

M.D Rusudan Peikrishvili

M.D Juliette Guibert

Ph.D Jean Bouyer

Long-term outcome of parenthood project during IVF and after discontinuation of unsuccessful IVF

Keywords: in vitro fertilization, follow-up study, treatment outcome, pregnancy, adoption

Objective: To explore the long-term outcome of patients who began IVF treatment by considering not only treatment outcome in the center but also the parenthood project outcome after discontinuation of unsuccessful IVF. Design: Retrospective cohort follow-up study. Setting: Two French IVF centers. Patient(s): 724 patients who began IVF treatment in 1998. Intervention(s): Postal and phone contacts with unsuccessful IVF patients. Main Outcome Measure(s): Long-term outcome of parenthood project.

Result(s).

Of the 724 patients, a minimum of 53% and a maximum of 81% finally succeeded in their parenthood project during or after IVF treatment (depending on the hypotheses that the 204 patients not contacted either failed or succeeded in their parenthood project). An intermediate hypothesis gave an estimation of 66% of patients finally succeeding in having a child (40% during IVF treatment in the center and 26% after). Achievement of the parenthood project after IVF discontinuation was mainly due to adoption of a child (46%) or a birth following a spontaneous pregnancy (42%).

Conclusion(s):

Unsuccessful patients should not lose hope, as nearly half may subsequently succeed in having a child.

INTRODUCTION

Infertility is an important public health issue, as 9% of couples in developed countries do not conceive within 12 months of attempting pregnancy and it is estimated that 56% of infertile couples seek medical help [START_REF] Boivin | International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care[END_REF]. In response to infertility, assisted reproductive techniques (ART) have been developing rapidly in the last thirty years.

In the European ART report for the year 2003, 15 countries (including Denmark, France, Germany, Norway, Sweden, UK) exhaustively reported their activity, finding a rate of 1,022 ART cycles per million inhabitants [START_REF] Andersen | Assisted reproductive technology in Europe, 2003. Results generated from European registers by ESHRE[END_REF].

In spite of a clear increase in the number of ART cycles performed worldwide, these treatments remain an emotional and physical burden for the patient and her male partner [START_REF] Eugster | Psychological aspects of in vitro fertilization: a review[END_REF][START_REF] Schmidt | Psychosocial burden of infertility and assisted reproduction[END_REF] and drop-out rates are high. In countries where the state subsidizes three IVF cycles, it has been observed that drop-out rates before the third cycle are as high as 62% in the Netherlands [START_REF] Land | Patient dropout in an assisted reproductive technology program: implications for pregnancy rates[END_REF] and 65% in Sweden [START_REF] Olivius | Cumulative probability of live birth after three in vitro fertilization/intracytoplasmic sperm injection cycles[END_REF]. In the United Kingdom where IVF treatment is often financed by the couples themselves, it has been observed than 64% of couples discontinued treatment after only one unsuccessful IVF cycle [START_REF] Sharma | Factors influencing the cumulative conception rate and discontinuation of in vitro fertilization treatment for infertility[END_REF]. These high drop-out rates raise several questions. The first is the reason for discontinuation. The few papers having addressed this issue highlighted the importance of predictors of failure and the psychological burden of treatment, especially when IVF cycles are state-subsidized [START_REF] Sharma | Factors influencing the cumulative conception rate and discontinuation of in vitro fertilization treatment for infertility[END_REF][START_REF] Domar | Impact of psychological factors on dropout rates in insured infertility patients[END_REF][START_REF] Olivius | Why do couples discontinue in vitro fertilization treatment? a cohort study[END_REF][START_REF] Penzias | When and why does the dream die? or does it?[END_REF]. A second question is to how to take drop-outs into account when estimating the probability that IVF couples will finally have a child. In evaluation of the efficacy of IVF, this issue has been addressed by advancing hypotheses on the couples' probability of success if they had continued IVF treatment [START_REF] Land | Patient dropout in an assisted reproductive technology program: implications for pregnancy rates[END_REF][START_REF] Olivius | Cumulative probability of live birth after three in vitro fertilization/intracytoplasmic sperm injection cycles[END_REF][START_REF] Soullier | Estimating the success of an in vitro fertilization programme using multiple imputation[END_REF][START_REF] Stolwijk | A more realistic approach to the cumulative pregnancy rate after in-vitro fertilization[END_REF]. Another approach would be to examine the real chance of becoming parents for patients starting IVF, either during the treatment itself or after drop-out. In particular, it would be of great interest to investigate whether drop-out patients finally succeed in having a child, possibly by transition of their parenthood project toward adoption.

In order to address this issue, we followed patients from the beginning of IVF treatment and examined not only treatment outcome in the center but also the longterm parenthood project outcome after discontinuation of unsuccessful treatment.

MATERIALS AND METHODS

Design and setting

Patients were from two French IVF centers in Paris (Cochin Hospital) and in Clermont-Ferrand, a medium-sized city in central France. All patients who had their first oocyte retrieval in one of these centers in 1998 were included. The study received approval from the French Data Protection Authority (CNIL) in September 2005 (authorization number 05-1334).

In May 2006, a postal questionnaire was sent to women who had discontinued IVF treatment without achieving a live birth. Treatment discontinuation was defined as no oocyte retrieval, no fresh or frozen embryo transfer, and no IVF pregnancy in the inclusion center within two years preceding the mailing. A postal questionnaire was mailed to the address recorded at the time of treatment. If the mail was returned because the patient was not known at that address, we searched for an up-to-date address in the hospital general addresses database and in the French national phone directory. In September 2006, a second postal mail including a refusal form was sent to non-respondents. If we received no answer to the two postal mails, we tried to contact the patient by telephone between November and December 2006 in order to complete a very short phone questionnaire.

Information Collected

Information on IVF attempts in the inclusion center was collected from medical records: infertility diagnosis, successive oocyte retrievals undergone by the patients in the center (conventional IVF or ICSI), outcomes of IVF pregnancies obtained either by fresh or frozen embryo transfers.

The postal questionnaire covered a wide range of topics: sociodemographic characteristics, fertility before 1998, treatments undergone before 1998, patient's feelings about IVF treatment, IVF discontinuation, patient's history since the end of IVF treatment in the inclusion center, in particular IVF attempts in another center, other medical treatments, spontaneous pregnancies, adoption procedures, bringing up other children, opinion on IVF treatment, consequences of IVF treatment. The short phone questionnaire was restricted to patient's profession, success in having a child after the end of IVF treatment in the inclusion center, spontaneous pregnancies, and psychological consequences of IVF treatment.

Statistical Methods

Data were analyzed using SAS software (9.0 version). Chi-square tests or Fisher exact tests were used for comparing percentages. Crude and adjusted odds ratios (OR) and 95% confidence intervals (CI) were estimated by logistic regression.

RESULTS

Of the 724 patients who began IVF treatment in the Cochin or Clermont-Ferrand centers in 1998, 293 (40%) patients obtained a live birth in the inclusion center, 421 (58%) patients stopped treatment in the center without a live birth and 10 (2%) patients were either still treated in the center or had an incomplete medical record which made it impossible to determine their treatment outcome (Table 1).

Of the 421 patients who discontinued IVF treatment, 420 were included in the long-term survey (one patient was deceased). Of these, 231 (55%) patients were treated at Cochin and 189 (45%) at Clermont-Ferrand. One hundred twenty-three (29%) patients completed the postal questionnaire, 93 (22%) patients responded to the brief phone questionnaire and 204 (49%) patients were non-respondents. Most non-respondents (n = 172, 84%) were patients who could not be either traced or contacted by telephone.

In order to investigate potential bias due to non-response, we compared the three categories of women included in the long-term survey (Table 2): women who responded to the postal questionnaire, women who responded to the phone questionnaire and women who did not respond. They did not significantly differ in age and infertility diagnosis. Non-respondents were more frequent in the Cochin center, if less than 4 oocyte retrievals had been performed and if a long time (6 years or more) had elapsed since discontinuation of IVF in the inclusion center. Among respondents, those who had answered the postal questionnaire were compared with those who answered the short phone questionnaire. We found no difference in patient's profession. Self-reported psychological difficulties still persisting after discontinuation of unsuccessful IVF treatment were nearly twice as high among respondents to the short phone questionnaire (26% versus 14%, p = 0.03). A slightly higher percentage of postal questionnaire respondents than phone questionnaire respondents had achieved their parenthood project (46% versus 41%) or had a spontaneous pregnancy (21% versus 16%). However, the differences were not statistically significant.

Taken as a whole, of the 216 respondents to the postal or phone questionnaire, 95 (44%) declared they had achieved their parenthood project since discontinuing unsuccessful IVF treatment in the inclusion center, including 41 (19%) who had conceived spontaneously. For the 204 patients who did not participate in the long-term survey (non-respondents), various hypotheses can be advanced as to their long-term outcome.

Hypothesis n°1 (the extremely pessimistic hypothesis): none of the 204 patients who did not participate in the long-term survey had achieved her parenthood project. This would mean that only 95 patients achieved their parenthood project, representing 23% of the patients who discontinued unsuccessful IVF treatment and 13% of the initial 724 patients who began IVF treatment in 1998.

Hypothesis n°2 (the intermediate hypothesis): the 204 non-participant patients

achieved their parenthood project in the same proportion as respondents to the postal or phone questionnaire (44%). This would give 185 (=95+204x0.44) patients who achieved their parenthood project after discontinuation of unsuccessful IVF treatment, representing 44% of the unsuccessful IVF patients and 26% of the initial patients.

Hypothesis n°3 (the extremely optimistic hypothesis): all the 204 nonparticipant patients achieved their parenthood project. This would give 299 (=95+204) patients who achieved their parenthood project after discontinuation of unsuccessful IVF treatment, representing 71% of the unsuccessful IVF patients and 41% of the initial patients.

Adding the 40% of patients who had a live birth during IVF treatment, we estimated that the proportion of patients who finally succeeded in their parenthood project during or after IVF treatment was 53% (pessimistic hypothesis), 66% (intermediate hypothesis) and 81% (optimistic hypothesis).

Among respondents to the postal questionnaire (n = 123), we were able to explore long-term outcome in greater depth (Table 3). 46% had achieved their parenthood project, 23% had not but were still trying to have a child and 31% had given up their parenthood project. 17% no longer lived with their male partner of 1998. After discontinuing treatment in their IVF center, patients took different steps to achieve their parenthood project (sometimes with a new male partner): adoption procedure (42%), IVF treatment in another center (20%), or other medical treatment, in particular artificial insemination with husband or donor sperm (7%). Among women who achieved their parenthood project after discontinuing unsuccessful treatment, two types of parenthood project achievement predominated: adoption (46%) and spontaneous pregnancy (42%). Births after medical treatment (including IVF) appeared much less frequent (12%). One woman succeeded in two different ways: she first adopted a child and then had a spontaneous pregnancy.

Factors associated with a successful parenthood project are shown in Table 4.

In univariate analysis, six variables were significantly associated with parenthood project achievement. Success was more frequent if the patient was aged less than 35 years at the first oocyte retrieval, if her educational level was medium or high, if she had no child when beginning IVF treatment in the inclusion center in 1998, if duration of infertility in 1998 was less than 5 years, if the decision to discontinue IVF treatment was not voluntary (for example because the treatment was no longer reimbursed) and if in 2006 the patient still lived with the same male partner as in 1998. In multivariate analysis, only three variables remained significant (having no child when beginning IVF treatment, duration of infertility and still living with the same partner).

DISCUSSION

Among 724 patients undergoing a first oocyte retrieval in 1998 in one of the two French IVF centers, we estimated, using an intermediate hypothesis, that 66% of patients finally succeeded in having a child during or after IVF treatment. If we considered extremely pessimistic and optimistic hypotheses (all patients lost to follow-up failed or all succeeded), we concluded that a minimum of 53% and a maximum of 81% of patients finally succeeded in their parenthood project. These percentages could continue to rise as more time elapsed since treatment initiation. Indeed, patients could still be attempting to achieve their parenthood project at the time of the survey (as were 23% of respondents to the postal questionnaire).

According to the intermediate hypothesis (66% of patients finally succeeding in having a child during or after IVF treatment), the parenthood project was achieved by IVF treatment in the inclusion center (40%), by adoption of a child (12%), spontaneous pregnancy leading to a live birth (11%) or other medical treatment or IVF treatment in another center after discontinuation of unsuccessful treatment in the inclusion center (3%).

Our results obtained in a French population exhibited the same trends as those observed recently in a Norwegian population. Analyzing long-term outcome of 108 patients referred to one Norwegian IVF center in 1996 and interviewed 10 years later, Sundby et al. found an overall success probability of 77%, including 47% of patients who had a child during IVF treatment, 15% who adopted a child and 15% who had a child following a spontaneous pregnancy [START_REF] Sundby | Consequences of IVF among women: 10 years post-treatment[END_REF]. In this Norwegian study, the authors also reported that 13% of patients were bringing up a child (10% as a foster parent and 3% as a stepmother).

In our study, we had information on only half of the patients having discontinued IVF without a live birth in the inclusion center. However this low response rate was not linked to refusal to participate: nearly all patients agreed to respond at least to a very short phone questionnaire and 84% of non-respondents could in fact not be contacted because they had moved since treatment. This is reassuring inasmuch as a high refusal rate often introduces selection bias. Moreover, in our study, non-response was clearly linked with variables that could be associated with a higher risk of a change of address: treatment in the Paris region (Cochin center) where the population is more mobile than in other regions of France (such as Clermont-Ferrand), a longer time elapsing since IVF discontinuation and having less than four oocyte retrievals, which is linked with time elapsed since IVF. The rate of follow-up loss observed in our study (49%) was close to that (42%) observed in a Brazilian study based on phone interviews of 92 couples who had unsuccessfully undergone one or more IVF cycles in an IVF center at least 3 years before the start of the study [START_REF] Filetto | Long-term follow-up of women and men after unsuccessful IVF[END_REF]. Such a high rate of follow-up loss in our study was due to the long time elapsing between IVF treatment discontinuation and our survey, more than 6 years for 89% of non-respondents versus 79-82% of respondents (p=0.04). Such a long period was necessary to investigate long-term outcome (especially adoption, a procedure which requires several years). Another methodological approach could be to consider active follow-up of a cohort of patients. However, this approach is very time-consuming and costly and could finally be inefficient. Firstly, the topic of our survey is a very sensitive one for patients and questionnaires repeated over time could be difficult to administer. Secondly, loss of follow-up in a cohort is commonly as high as 40-50% after 6 years. For example, in a French cohort on contraception, of the 2,863 women who agreed in 2000 to participate in a yearly phone contact, 33% no longer participated only two years later [START_REF] Razafindratsima | Attrition in the COCON cohort between 2000 and 2002[END_REF] and about 20% of UK families who participated in the first sweep of the Millennium Cohort Study did not respond to the second sweep just two years later [START_REF] Plewis | Millennium Cohort Study: Technical Report on Response[END_REF]. When investigating long-term parenthood projects among unsuccessful IVF treatment patients, which is a very sensitive topic for those who have not yet succeeded, one could expect an even higher rate of follow-up loss if carrying out repeated distressing interrogations in a prospective cohort.

A high rate of non-response could lead to selection bias in our study if reasons for moving were related to the patient's probability of achieving her parenthood project. We first considered that the change of residence could be related to separation of the couple, which was a major predictive factor of failure to have a child after IVF treatment discontinuation among respondents. So, non-respondents could have a lower chance of success than respondents. We also considered that, on the contrary, change of residence could be related to the arrival of a child in the family and the need for an additional bedroom [START_REF] Courgeau | Estimation des migrations internes de la période 1990-1999 et comparaison avec celles des périodes antérieures[END_REF]. In view of these two contradictory assumptions, we were unable to conclude on the direction selection bias would go, whether it would imply over-or under-estimation of the probability of finally having a child. Finally, two methods of interviewing patients were used in the study (postal questionnaire and phone interview). We do not believe that this would induce differential bias in factual information such as the birth or adoption of a child.

However, it could have induced differences for less factual information such as psychological well-being. In Table 2, we observed that patients responding to the short phone questionnaire were twice as likely to declare psychological problems than respondents to the postal questionnaire. Psychological difficulties could be the reason why these patients did not answer the postal questionnaire, but we could not exclude that this partly reflected differences linked to the method of interview (phone or postal questionnaire).

In our multivariate analysis, achievement of the parenthood project after discontinuation of unsuccessful IVF treatment in the inclusion center was positively linked to two initial characteristics of the patient: not having a child at the beginning of IVF treatment and infertility duration of less than 5 years in 1998. Both could reflect patients' motivation and determination to succeed in having a child (even by adoption) and/or better fertility chances leading to spontaneous pregnancies (patients with shorter infertility duration possibly being more fertile and also younger). Two other variables could be interpreted in the same way: young age of the patient at the first oocyte retrieval and the fact that discontinuation of IVF treatment was not a voluntary decision by the couple. However, these two variables were significant only in univariate analysis. Moreover, patients separated from their male partner appeared to have less chance of finally having a child, probably because separation could have put an end to the parenthood project and/or because the patient was then alone. This suggests that the long-term outcome of a parenthood project may vary from one country to another according to the frequency of separations. For example, in France, the divorce rate was 42.4% in 2003, while it was higher (56.1%) in Belgium and very much lower in Spain (14.5%) [START_REF] Sardon | Recent demographic trends in developed countries[END_REF]. Similar research on the long-term outcome of parenthood project should be carried out in other countries in order to verify that the observations made in French couples can be generalized.

Our results showed the importance of spontaneous pregnancy and of adoption in achieving the parenthood project after discontinuation of unsuccessful IVF treatment, whereas only a small proportion of patients became parents following another medical treatment. Surprisingly, although 20% of patients followed another IVF program in another center, only 21% of these patients had a child in this way. In our survey, 19% of respondents reported that they had spontaneously conceived since discontinuation of IVF. This proportion is similar to those reported in two studies analyzing spontaneous pregnancy rate among IVF patients who had a live birth after treatment: a Japanese study observed that 18% of 142 patients conceived spontaneously within 5 years after the birth of their IVF child [START_REF] Shimizu | Spontaneous conception after the birth of infants conceived through in vitro fertilization treatment[END_REF] and an Irish study found that 21% of 513 patients conceived spontaneously after an IVF birth [START_REF] Hennelly | Spontaneous conception after a successful attempt at in vitro fertilization/intracytoplasmic sperm injection[END_REF]. In a Belgian study, spontaneous pregnancies were analyzed among 200 patients discontinuing ICSI [START_REF] Osmanagaoglu | Spontaneous pregnancies in couples who discontinued intracytoplasmic sperm injection treatment: a 5-year follow-up study[END_REF]. The authors observed a slightly lower rate of spontaneous pregnancy, 12-13%, but the time since treatment discontinuation was shorter (3 years).

Our long-term survey showed that adoption and spontaneous conception are important issues to consider when dealing with the long-term outcome of parenthood projects among couples beginning an IVF program. It would be very interesting to examine in greater depth which patients finally conceived spontaneously and which patients finally adopted a child. This approach was not possible in our study because the sample size was too small, so more research would be needed on this topic.

In conclusion, we gained new insight for IVF patients by taking a long-term view rather than considering their parenthood project only in the IVF center. While for 40% of patients IVF treatment resulted in a successful pregnancy, unsuccessful patients should not lose hope, as nearly half of them may subsequently have a child.

Unknown treatment outcome b n (%) 4

a: We considered that a patient was still in treatment in the inclusion center if she had an oocyte retrieval or a fresh or frozen embryo transfer or an IVF pregnancy in the center in the two years before our mailed questionnaire in May 2006.

b: Outcome of IVF treatment was unknown for 4 patients in the Cochin center because the information was missing from the medical records. b: Low level of education was defined as < baccalauréat; medium-high level was defined as ≥ baccalauréat (completion of secondary education). c: Main reasons for considering that IVF discontinuation was not a choice but was involuntary were that the treatment was no longer reimbursed or that the patient was too old to be included in an IVF treatment program.

  = 107 patients of 123 with complete information for the 12 variables entered in the multivariate model.

TABLE 1 . Treatment outcomes among the 724 patients who began IVF treatment in 1998 in one of the two IVF centers.

 1

	IVF center
	Cochin	Clermont-	Total
		Ferrand
	392	332	724

TABLE 2 . Characteristics (%) of postal questionnaire respondents, short phone questionnaire respondents, and non-respondents

 2 (n = 420 a patients included in the long-term survey). Time elapsed in 2006 since discontinuation of unsuccessful IVF treatment in the inclusion center was from 3 to 8 years. Six years was the first percentile (median: 7 years, 3 rd percentile: 8 years). e: NA = not applicable: this information was not available for non-respondents.

	Postal questionnaire respondents	Short phone questionnaire respondents	Non-respondents b	p c
		(n = 123)	(n = 93)	(n = 204)
	IVF center				0.01
	Cochin	44	60	59
	Clermont-Ferrand	56	40	41
	Patient age in 1998				0.74
	< 35 years	59	55	59
	 35 years	41	45	41
	Infertility diagnosis based on medical records		0.70
	Female	43	38	33
	Male	27	30	28
	Female and male	17	18	22
	Unexplained	13	14	16
	Number of oocyte retrievals				0.02
	< 4	82	73	86
	 4	18	27	14
	Time elapsed in 2006 since IVF discontinuation
	in the inclusion IVF center d				0.04
	< 6 years	21	18	11
	≥ 6 years	79	82	89
	Patient with a managerial profession			0.88
	No	87	88	NA e
	Yes	13	12	NA e
	Achievement of parenthood project since IVF discontinuation	0.42
	No	54	59	NA e
	Yes	46	41	NA e
	Spontaneous pregnancy since IVF discontinuation		0.36
	No	79	84	NA e
	Yes	21	16	NA e
	Still psychologically affected by IVF treatment		0.03
	No	86	74	NA e
	Yes	14	26	NA e
	a: Of the 421 patients who discontinued unsuccessful IVF treatment, one was deceased.

b: Non-respondents include patients lost to follow-up (n = 178) and patients refusing to answer (n = 26). c: p value of the chi-squared or Fisher exact test. d:

TABLE 3 . Long-term parenthood project outcome after discontinuation of unsuccessful IVF treatment in the inclusion center among the 123 respondents to the postal questionnaire in the long-term survey. Long-term outcome of the patient's parenthood project

 3 If the patient had more than one child, we only considered the first, so the sum of column percentages is 100%.

	Succeeded in her parenthood project	46%
	Project continuing	23%
	Project abandoned	31%
	Situation of the couple since IVF discontinuation
	Still living together	83%
	Separated	17%
	Steps taken to achieve (successfully or not) the parenthood
	project a
	IVF treatment in another center	20%
	Medical treatment other than IVF	7%
	Start of adoption procedure	42%
	For patients with a successful parenthood project (n = 57),
	method by which it had been achieved b
	IVF treatment in another center	7%
	Other medical treatment (including AI with donor	5%
	sperm)
	Adoption	46%
	Spontaneous pregnancy leading to a live birth	42%

a: One patient may have taken none, one or more than one steps, so the sum of percentages is not 100%.

b:

TABLE 4 . Factors associated with achievement of parenthood project after discontinuation of unsuccessful IVF treatment in the inclusion center among the 123 respondents to the postal questionnaire.

 4

	Patients achieving their
	parenthood project	Crude OR	Adjusted OR
	n	%	[95% CI]	[95% CI] a
	IVF center
	Clermont-Ferrand	69	54	1.00	1.00
	Cochin	54	37	0.51 [0.25 -1.05]	0.84 [0.28 -2.49]
	Patient age at first oocyte retrieval
	< 35 years	73	58	1.00	1.00
	≥ 35 years	50	30	0.32 [0.15 -0.68]	0.44 [0.13 -1.50]
	Educational level of the patient b
	Low	55	36	1.00	1.00
	Medium or high	66	56	2.23 [1.07 -4.65]	2.05 [0.67 -6.27]
	Patient fertility in 1998 when beginning IVF treatment
	No child	91	59	1.00	1.00
	At least one child	29	7	0.05 [0.01 -0.23]	0.05 [0.01 -0.26]
	Infertility duration in 1998 when beginning IVF treatment
	< 5 years	58	57	1.00	1.00
	≥ 5 years	61	38	0.46 [0.22 -0.95]	0.22 [0.06 -0.78]
	Infertility diagnosis based on medical records
	Female	53	49	1.00	1.00
	Male	33	61	1.60 [0.66 -3.86]	0.80 [0.20 -3.15]
	Female and male	21	29	0.42 [0.14 -1.23]	0.33 [0.07 -1.45]
	Unexplained	16	31	0.47 [0.14 -1.55]	0.44 [0.08 -2.49]

Opinion expressed by the patient in 2006 on the inclusion IVF center

	Satisfied or very satisfied	86	42	1.00	1.00
	Unsatisfied or very	36	58	1.94 [0.88 -4.28]	1.89 [0.50 -7.10]
	unsatisfied

Number of oocyte retrieval performed in the inclusion IVF center

	< 4	101	46	1.00	1.00
	≥ 4	22	50	1.20 [0.48 -3.01]	1.01 [0.20 -5.11]
	Time elapsed since discontinuation of IVF treatment
	< 6 years	26	50	1.00	1.00
	≥ 6 years	97	45	0.83 [0.35 -1.97]	2.38 [0.43 -13.0]
	Decision to discontinue IVF treatment
	Couple's decision	76	41	1.00	1.00
	Medical staff decision	22	32	0.68 [0.25 -1.85]	2.33 [0.46 -11.9]
	Not a choice, obligation c	22	82	6.53 [2.02 -21.2]	3.37 [0.65 -17.5]
	Situation of the couple since IVF discontinuation
	Still living together	102	52	1.00	1.00
	Separated	21	19	0.22 [0.07 -0.69]	0.10 [0.02 -0.53]
	Patient's psychological difficulties due to IVF treatment (self-reported in 2006)
	No difficulties	35	43	1.00	1.00
	Overcoming difficulties	66	55	1.60 [0.70 -3.66]	1.09 [0.31 -3.81]
	Still affected	16	38	0.80 [0.24 -2.69]	0.29 [0.05 -1.58]

Conflict of interest: none declared

Running title: Long-term outcome of IVF patients

Capsule: 66% of IVF patients may finally succeed in having a child: 40% during IVF treatment and 26% after discontinuing treatment (by adoption or spontaneous pregnancy).


Giovanni Busco

Azadeh Valinataj Omran

Loïck Ridou

Jean-Michel Pouvesle

Eric Robert

Catherine Grillon

Fluorescein-agarose gel as a tissue model to visualize and measure local CAP-induced acidification

Keywords: agarose gel model, pH measurement, fluorescein, plasma jet

Cold Atmospheric Plasmas (CAPs) have been shown to lower the pH of the treated target. CAP-induced acidification can have beneficial effects on the treated tissue (e.g. disinfection and healing). Conversely a tissue hyper acidification can lead to permanent damages with dramatic consequences. Before starting in vivo plasma treatments, preliminary tests should be carried in order to tune the acidifying effect induced by CAP exposition. Here we propose a tissue model that can be a useful tool to visualize and measure pH changes induced by plasma treatment.

Introduction

Once in contact with the atmosphere and water, Cold Atmospheric Plasmas (CAPs) initiate complex chemical reactions that lead to Reactive Oxygen and Nitrogen Species (RONS) generation. The produced RONS are mostly responsible for the biological effects observed during and after CAP treatment. Besides their oxidative effect, some CAP-generated species possess also an acidic character. The acidifying effect induced by CAP on treated liquids is widely known [START_REF] Brisset | Acidity control of plasmachemical oxidation: applications to dye removal, urban waste abatement and microbial inactivation[END_REF][START_REF] Oehmigen | The Role of Acidification for Antimicrobial Activity of Atmospheric Pressure Plasma in Liquids[END_REF]. While measuring the pH drop in liquids is relatively easy, the evaluation of the pH on solid surface is more complex. Flat pH-meters permit today to measure the pH on solid surfaces and organs like the skin. However, once applied to the solid target, these flat pH-meters give an average value of the pH on the covered surface. The relatively large surface of the pH-meter electrode does not permit to appreciate pH distribution, especially during a focalized CAP treatment where RONS are concentrated on a small surface. In this work we took advantage of the fluorescein fluorophore whose fluorescence is pH sensitive [START_REF] Emmart | Observations on the absorption spectra of fluorescein, fluorescein derivatives and conjugates[END_REF][4][START_REF] Martin | Sodium fluorescein as a retinal pH indicator?[END_REF] to study the CAP induced acidification in agarose tissue models.

Method

In this work we used a modified protocol of our recently published method [START_REF] Busco | Cold atmospheric plasma-induced acidification of tissue surface: visualization and quantification using agarose gel models[END_REF]. Briefly a buffered physiological solution (130mM NaCl, 5mM KCl, 2mM MgCl2, 5.5mM HEPES) was prepared at 6 different pH ranging from 7.4 to 2.5. Fluorescein sodium salt (50µM) and DNA grade agarose (0.02g/ml) were added to each solution and then agarose was gently melted in a microwave. Gels at different pH were poured in 6well plates in order to obtain a 2 mm thick gel/well. Gels were let to solidify and then kept at 4°C. Before plasma treatment plates were allowed to equilibrate at room temperature. Gel fluorescence was then imaged using the Typhoon™ FLA 9500 biomolecular imager (GE Healthcare Life Science) using an excitation laser at 473 nm and a Y520 emission filter with a wavelength range ⩾ 520 nm. Immediately after the scan, the 6-well plate was exposed to CAP treatments. We treated the gel at initial pH of 7.4 (as generic organ model) and the gel at initial pH of 5.5 (as skin model). Plasma treatment was performed using a Plasma Gun [START_REF] Robert | Characterization of pulsed atmospheric-pressure plasma streams (PAPS) generated by a plasma gun[END_REF] with a negative polarity high voltage pulses having a peak amplitude of -14 kV, full width at half maximum of 2 μs duration. Gels were exposed for 5 min to the thin jet produced at the tip of a thin tapered capillary (1.5 mm internal diameter). Helium was used as feeding gas at a flow rate of 0.5 SLM. After the treatment, the plate was scanned again to image the changes in the fluorescence induced by CAP exposure. 8-bit grey scale images were imported in ImageJ for data analysis. The scan of the 6well plate before CAP treatment was used to calibrate the fluorescence signal vs the 6 known pH. A calibration curve was thus obtained and used to measure the CAP-induced pH drop. To better visualize the pH variations, grey scale images were converted in spectrum pseudocolor in ImageJ. pH analysis on the plasma treated surface was done by tracing a straight line passing by the middle of the acidified area and measured values were plotted in a graph (figure 2).

Results

Immediately after the CAP treatment, acidified area appears as a darker spot on the green agarose gel. Fluorescein fluorescence is gradually quenched when the pH in the medium decreases. To better visualize the CAP-induced acidification, the 8-bit grey scale images were converted in pseudocolors (figure 1). The spatial pH distribution, measured by tracing a straight line passing through the CAP-exposed surface showed a pH decrease of one unit in the generic organ model, the generic organ model and a slightly higher decrease in the skin model (figure 2). The lower acidification observed in the skin model could be attributed to the HEPES buffering activity having its optimum in the pH range between 8.2 and 6.8.

Conclusions

In this work we propose a novel technique useful to evaluate and measure the CAP-induced acidification on agarose-gel tissue models. Agarose-gels with initial pH of 7.4 or 5.5, mimicking respectively a generic organ or the outer skin pH, were used to evaluate the acidifying effect of plasma treatment. These models can be useful during CAP treatment planning, especially when a focalized treatment should be administered. Controlling the local induced acidification can help to avoid hyper-acidification that can cause chemical burn on the CAP exposed tissue.

Figure 1 .

 1 Figure 1. Visualization of the acidification on the fluorescent agarose gel models. To better visualize the acidified area, 8-bit images were converted in spectrum pseudocolor in ImageJ

Figure 2 .

 2 Figure 2. Plotted graphs of pH measured along a manually traced straight line passing through the CAP-exposed surface