Marion Rouge
email: marion.rouge@unicaen.fr

Razan Elkhatib

Christelle Delalande

Juliette Cognié

Fabrice Reigner

Philippe Barrière

Stefan Deleuze

Matthieu Cousty

Florence Legendre

Philippe Galéra

Vincent Hanoux

Hélène Bouraïma-Lelong

Investigation of equine testis contribution to vitamin D bioactivation

Introduction

Vitamin D is a key regulator of cell differentiation and proliferation, calcium homeostasis and bone metabolism [START_REF] Bouillon | Structure-Function Relationships in the Vitamin D Endocrine System[END_REF][START_REF] Fleet | Serum Metabolite Profiles and Target Tissue Gene Expression Define the Effect of Cholecalciferol Intake on Calcium Metabolism in Rats and Mice[END_REF][START_REF] Holick | Vitamin D deficiency: a worldwide problem with health consequences[END_REF]. Indeed, vitamin D regulates the intestinal absorption of calcium, renal reabsorption and bone metabolism thus allowing the maintenance of the calcium concentration for the calcium-dependent physiological functions [START_REF] Bouillon | Vitamin D and Human Health: Lessons from Vitamin D Receptor Null Mice[END_REF][START_REF] Haussler | Molecular Mechanisms of Vitamin D Action[END_REF]. In humans, the main source of vitamin D is synthesized in the skin where UV-B radiations emitted by the sun's rays convert cutaneous 7-dehydrocholesterol into cholecalciferol, namely the inactive form of vitamin D.

Then, the biological form of vitamin D results from two successive hydroxylation reactions [START_REF] Prosser | Enzymes involved in the activation and inactivation of vitamin D[END_REF][START_REF] Gray | Vitamin D metabolism: the role of kidney tissue[END_REF]. To this end, cholecalciferol is transported to the liver where the first hydroxylation reaction occurs, mainly mediated by CYP2R1 and supported by CYP27A1. The newly formed 25-hydroxyvitamin D3 will be transported to the kidneys where the second hydroxylation reaction mediated by CYP27B1 occurs [START_REF] Morris | Autocrine and paracrine actions of vitamin D[END_REF]. A gene mutation of CYP2R1 or CYP27B1 results in low levels of 1α,25-Dihydroxyvitamin D3 (1,25(OH)2 vitamin D3) as well as defective calcium homeostasis leading to bone damages [START_REF] Kato | Editorial: Vitamin D 1α-Hydroxylase Knockout Mice as a Hereditary Rickets Animal Model[END_REF][START_REF] Kitanaka | Inactivating Mutations in the 25-Hydroxyvitamin D 3 1α-Hydroxylase Gene in Patients with Pseudovitamin D-Deficiency Rickets[END_REF]. The biologically active form of vitamin D, 1α,25(OH)2 vitamin D3 generates its transcriptomic effects by binding to its receptor VDR (Vitamin D Receptor) in target tissues until its inactivation by CYP24A1 enzyme [START_REF] Prosser | Enzymes involved in the activation and inactivation of vitamin D[END_REF].

The expression of vitamin D metabolizing enzymes (CYP2R1, CYP27A1, CYP27B1 and CYP24A1), was previously described as being limited exclusively to the liver and kidneys. However, the expression of these enzymes, as well as VDR, have been evidenced in a variety of tissues including, male reproductive organs [START_REF] Choudhary | Expression patterns of mouse and human CYP orthologs (families 1-4) during development and in different adult tissues[END_REF][START_REF] Tirabassi | Association between vitamin D and sperm parameters: clinical evidence[END_REF][START_REF] De Angelis | The role of vitamin D in male fertility: a focus on the testis[END_REF]. Indeed, in humans, these enzymes and VDR are expressed in the testis, epididymis, seminal vesicles and prostate [START_REF] Aquila | Human male gamete endocrinology: 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) regulates different aspects of human sperm biology and metabolism[END_REF][START_REF] Blomberg Jensen | Vitamin D receptor and vitamin D metabolizing enzymes are expressed in the human male reproductive tract[END_REF][START_REF] Choudhary | Expression patterns of mouse and human CYP orthologs (families 1-4) during development and in different adult tissues[END_REF][START_REF] Corbett | Vitamin D receptor found in human sperm[END_REF]. In the human testis, all enzymes and the receptor are co-located in Leydig cells [START_REF] Blomberg Jensen | Vitamin D receptor and vitamin D metabolizing enzymes are expressed in the human male reproductive tract[END_REF][START_REF] Foresta | Bone Mineral Density and Testicular Failure: Evidence for a Role of Vitamin D 25-Hydroxylase in Human Testis[END_REF], in spermatocytes, round and elongated spermatids and spermatozoa. Only, CYP27B1, CYP24A1 and VDR are expressed in spermatogonia [START_REF] Blomberg Jensen | Vitamin D is positively associated with sperm motility and increases intracellular calcium in human spermatozoa[END_REF], 2010). Moreover, the expression of vitamin D metabolizing enzymes in the male reproductive system has been reported in many species. In addition to being expressed in the human testis, the enzymatic expression has also been demonstrated in rodent testis [START_REF] Johnson | Immunohistochemical detection and distribution of the 1,25-dihydroxyvitamin D3 receptor in rat reproductive tissues[END_REF][START_REF] Mahmoudi | Distribution of Vitamin D Receptor and 1α-Hydroxylase in Male Mouse Reproductive Tract[END_REF], sheep [START_REF] Yao | Age-associated expression of vitamin D receptor and vitamin D-metabolizing enzymes in the male reproductive tract and sperm of Hu sheep[END_REF] and roosters [START_REF] Oliveira | Vitamin D3 and androgen receptors in testis and epididymal region of roosters (Gallus domesticus) as affected by epididymal lithiasis[END_REF]. In these species, enzymes have also been demonstrated in male reproductive tissues, in particular in the efferent ductules [START_REF] Oliveira | Vitamin D3 and androgen receptors in testis and epididymal region of roosters (Gallus domesticus) as affected by epididymal lithiasis[END_REF], the epididymis (caput, corpus and cauda) [START_REF] Dornas | Distribution of vitamin D3 receptor in the epididymal region of roosters (Gallus domesticus) is cell and segment speciWc[END_REF][START_REF] Johnson | Immunohistochemical detection and distribution of the 1,25-dihydroxyvitamin D3 receptor in rat reproductive tissues[END_REF][START_REF] Mahmoudi | Distribution of Vitamin D Receptor and 1α-Hydroxylase in Male Mouse Reproductive Tract[END_REF][START_REF] Oliveira | Vitamin D3 and androgen receptors in testis and epididymal region of roosters (Gallus domesticus) as affected by epididymal lithiasis[END_REF][START_REF] Yao | Age-associated expression of vitamin D receptor and vitamin D-metabolizing enzymes in the male reproductive tract and sperm of Hu sheep[END_REF], ductus deferens, epithelial cells of the seminal vesicles [START_REF] Johnson | Immunohistochemical detection and distribution of the 1,25-dihydroxyvitamin D3 receptor in rat reproductive tissues[END_REF][START_REF] Mahmoudi | Distribution of Vitamin D Receptor and 1α-Hydroxylase in Male Mouse Reproductive Tract[END_REF] and prostate [START_REF] Johnson | Immunohistochemical detection and distribution of the 1,25-dihydroxyvitamin D3 receptor in rat reproductive tissues[END_REF].

The existence of a link between the testis and cartilage or bone tissues has been demonstrated in humans [START_REF] Kato | Editorial: Vitamin D 1α-Hydroxylase Knockout Mice as a Hereditary Rickets Animal Model[END_REF] and rodents [START_REF] Dardenne | Targeted Inactivation of the 25-Hydroxyvitamin D3-1α-Hydroxylase Gene (CYP27B1) Creates an Animal Model of Pseudovitamin D-Deficiency Rickets*[END_REF]. In fact, men with hypogonadism had significantly lower vitamin D levels than healthy individuals [START_REF] Lee | Association of hypogonadism with vitamin D status: the European Male Ageing Study[END_REF][START_REF] Wang | Vitamin D is associated with testosterone and hypogonadism in Chinese men: Results from a cross-sectional SPECT-China study[END_REF]. Avitaminosis associated with hypogonadism resulted in an altered bone architecture and a low bone mineral density (BMD) which increased the risk of fractures [START_REF] Misra | Effects of hypogonadism on bone metabolism in female adolescents and young adults[END_REF]. Elevated vitamin D levels have also been shown to be associated with hypogonadism [START_REF] Lerchbaum | Serum vitamin D levels and hypogonadism in men[END_REF]. In addition, a decrease in vitamin D levels has been demonstrated in orchiectomized patients after a diagnosis of testicular cancer [START_REF] Foresta | Altered bone status in unilateral testicular cancer survivors: Role of CYP2R1 and its luteinizing hormone-dependency[END_REF][START_REF] Foresta | Testiculopathy and vitamin D insufficiency[END_REF][START_REF] Schepisi | Vitamin D status among long-term survivors of testicular cancer[END_REF]. Low levels of vitamin D resulted in altered BMD [START_REF] Foresta | Altered bone status in unilateral testicular cancer survivors: Role of CYP2R1 and its luteinizing hormone-dependency[END_REF]. These various dysfunctions have demonstrated a testicular contribution to the vitamin D bioactivation in humans and the impact of vitamin D on osteoarticular metabolism [START_REF] Kitanaka | Inactivating Mutations in the 25-Hydroxyvitamin D 3 1α-Hydroxylase Gene in Patients with Pseudovitamin D-Deficiency Rickets[END_REF]. To help reverse this testicular deficiency, vitamin D supplementation is necessary [START_REF] Bischoff-Ferrari | Prevention of Nonvertebral Fractures With Oral Vitamin D and Dose Dependency: A Meta-analysis of Randomized Controlled Trials[END_REF][START_REF] Trivedi | Effect of four monthly oral vitamin D3 (cholecalciferol) supplementation on fractures and mortality in men and women living in the community: randomised double blind controlled trial[END_REF].

Unlike other species, the horse is considered as an exception with regard to its calcium homeostasis, which is characterized by high plasma calcium level, high intestinal calcium absorption capacity, weaker renal reabsorption of calcium with a high urinary calcium excretion and a low plasma concentration of vitamin D metabolites [START_REF] Breidenbach | Peculiarities of vitamin D and of the calcium and phosphate homeostatic system in horses[END_REF][START_REF] Mäenpää | Serum Profiles of Vitamins A, E and D in Mares and Foals During Different Seasons[END_REF][START_REF] Schryver | The Site of Calcium Absorption in the Horse[END_REF][START_REF] Toribio | Essentials of Equine Renal and Urinary Tract Physiology[END_REF][START_REF] Toribio | Comparison of serum parathyroid hormone and ionized calcium and magnesium concentrations and fractional urinary clearance of calcium and phosphorus in healthy horses and horses with enterocolitis[END_REF]. In equine species, the kidney plays a more significant key role in calcium homeostasis than vitamine D [START_REF] Toribio | Essentials of Equine Renal and Urinary Tract Physiology[END_REF]. The low renal 1α-hydroxylase CYP27B1 activity leads to a low vitamin D level in this species [START_REF] Breidenbach | Peculiarities of vitamin D and of the calcium and phosphate homeostatic system in horses[END_REF][START_REF] Toribio | Essentials of Equine Renal and Urinary Tract Physiology[END_REF].

The presence of enzymes metabolizing vitamin D has not been documented in the testis of horses, but the castration of male horses is a regular surgical breeding practice aimed at removing the primary source of sex steroid hormones, especially testosterone which has an impact on behavior through its action on the brain [START_REF] Mhaouty-Kodja | Sexual Behavior: From Hormonal Regulation to Endocrine Disruption[END_REF]. This practice is carried out in a veterinary context without age recommendation to avoid side effects beyond the desired effects. In other words, this surgery is performed at any age: in foals, during the growth period or in adulthood. However, the consequences of castration on vitamin D metabolism have not been studied in equine species. Through this study, we sought to assess the contribution of the equine testis to the vitamin D bioactivation as it has been described in humans and some herbivores. To this end, the gene expression of vitamin D metabolizing enzymes in equine testis was studied. Subsequently, plasma levels of 1α,25(OH)2 vitamin D3 were quantified before and after castration in order to investigate the impact of castration on vitamin D levels in horses. For this purpose, three groups of castrated animals at different ages (1 week, 2.2 and 6.6 years old) and a non-castrated control group were studied.

Materials and methods

Materials and reagents

Animals and samples

The experiment was carried out with four horse groups (Equus Caballus) which were classified according to the age at which they were castrated: castrated neonates at birth (n=8; 1week), uncastrated neonates (n=8), castrated horses at post-pubertal stage (n=8, mean age: 2.2 years old) and castrated at adult stage (n=6; mean age: 6.6 years old) (Figure 1). For each group, a blood sample was collected before castration (P0). The blood samples from the male neonates' group with castration following birth and control group neonates without castration (Welsh ponies), were collected monthly over a period of 12 months. The foals from these two groups were born in the same period (June) and come from the "Animal Physiology Experimental Facility (PAO; INRAE Val de Loire, France). Regarding the group comprising animals castrated after puberty and the group comprising animals castrated at the adult stage, blood samples were collected at 3 and 6 months after castration. These animals come from the Saint-Michel de Livet veterinary clinic (Calvados, France).

Blood samples were collected into a tube with anticoagulant (EDTA), the plasma was separated by centrifugation at 3500 r.p.m. for 10 min at 4°C and stored at -80°C for subsequent hormone measurement.

Testes from 4 neonates (1 week), 5 post-pubertal (2.2 years old), 5 adults (6.6 years old) stallions were collected during routine castrations at the INRAE of Nouzilly (Indre et Loire, France) and the Centre Hospitalier Vétérinaire Équin de Livet (Calvados, France). One piece (1 cm 3 ), localized near the middle of the testis, was snapfrozen on dry ice and stored at -80°C for mRNA extraction.

RNA extraction and RT-PCR analysis

The total RNA was extracted from the testis of experimental herds with Nucleozol® reagent according to the manufacturer's instructions. Their concentration was quantified by absorbance at 260nm with NanoDrop® (Thermo Scientific). RNA quality was checked by measuring optical density at 260nm and 280nm. 1µg of total RNA was reverse transcribed into cDNA for 90min at 37°C with 100 IU of Moloney Murine Leukemia Virus Reverse Transcriptase, 25UI of RNasin, 0.5µg of random primers and 400µM of dNTP in a final volume of 40µL. In the absence of reverse transcriptase, a reaction was also included to check for genomic contamination. Negative control was used by adding RNase-free water instead of RNA. The resulting cDNA was used in PCRs with primer (Eurogentec, Belgium) designed to amplify specific regions of enzymes metabolizing vitamin D (Table 1). The reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified. The PCR mixture consisted of 8µL of cDNA, 1.5mM of MgCl2, 0.2mM of each dNTP, 1.25UI of Go Taq G2 Polymerase (Promega) and 16.6µM of each primer. The samples were amplified in Mastercycler (Eppendorf) as follows: one cycle of 2min at 95°C, followed by 30 cycles of 30s at 60°C, followed by an inactivation phase of 5min at 72°C. The PCR products were resolved onto agarose gels (1,5%) containing ethidium bromide (5%) and visualized under UV transillumination.

The reaction specificity was confirmed by PCR products sequencing (GENEWIZ, Germany).

Immunohistochemistry procedure

Tissue sections were deparaffinised in xylene and rehydrated through a series of graded ethanol 70%, 96%, 100%). To inhibit endogenous peroxidase activity, all sections were incubated in H2O2 3%/PBS for 10 minutes, following a sodium citrate buffer incubation (pH 6) in microwave oven at 90W for 10 minutes. The glass slides were incubated in Triton 0.2%/PBS for 20 minutes in order to permeabilize testicular sections. Then, all sections were blocked in 3% BSA / PBS solution for 20 minutes. The sections were exposed to the mouse anti-CYP27A1 antibody (Santa Cruz, USA) at 4°C overnight in a humid chamber followed by goat anti-mouse-HRP antibody (Cell signaling, USA) for 1h30 at room temperature. Then, the sections were visualized using the diaminobenzidine detection system (Sigma-Aldrich, USA). After counterstaining with Mayer's hematoxylin, testicular sections were dehydrated and covered with Eukitt. Negative control was processed using identical protocols except for the primary antibody was omitted.

ELISA

Testosterone and 1α,25(OH)2 vitamin D3 levels were measured using ELISA kits following the manufacturer's instructions (Enzo Life Science; detection limit: 3.9 -1000 pg/mL (Human reactivity) for testosterone and MyBioSource; detection limit: 31.2 -1000 pg/mL for 1,25(OH)2 vitamin D3). The samples were assayed in duplicate.

Statistical analysis

All data were presented as the mean ± SEM. A one-way ANOVA followed by a Tukey test was performed to compare the blood samples obtained before and after the castration of the experimental groups (adult, foals, castrated neonates and non-castrated neonates). A statistical analysis based on a t-test was carried out to compare the samples of both groups. Each sample was performed in duplicates. The differences were considered significant at P≤0.05.

Results

Testicular expression of CYP2R1, CYP27A1 and CYP27B1

To investigate the ability of testes to play a role in the vitamin D metabolism, the gene expression of CYP2R1, CYP27A1 and CYP27B1 was analyzed in the testis of neonates, post-pubertal and adult horses (Figure 2). A weak signal for CYP27A1 mRNA was detected in all three groups. The reaction specificity was confirmed by PCR products sequencing. Moreover, the expression of CYP27A1 was demonstrated in the interstitial tissue bu immunohistochemistry (Figure 3). No signal was detected for mRNAs of CYP2R1 and CYP27B1 regardless of the developmental stages studied. The absence of gene expression was confirmed by different pairs of primers, some of them were validated by other studies [START_REF] Azarpeykan | Evaluation and Comparison of Vitamin D Responsive Gene Expression in Ovine, Canine and Equine Kidney[END_REF].

Age-dependent circulating levels of 1α,25(OH)2 vitamin D3

The circulating levels of 1α,25(OH)2 vitamin D3 were quantified in plasma samples obtained from different groups formed for the study, before castration: neonates (1 week) (142,28 ± 5,71 pg/mL), post-pubertal horses (2.2 years old) (232,76 ± 11,82 pg/mL) and adults (6.6 years old) (194,01 ± 11,82 pg/mL) (Figure 4). A significant age-depending difference was observed. Indeed, in neonates, the 1α,25 (OH)2 vitamin D3 levels were significantly lower than in the post-pubertal and adult groups. On the other hand, no significant difference was observed between foals and adults.

1α,25(OH)2 vitamin D3 levels in neonates castrated or not castrated before puberty

The circulating 1α,25 (OH)2 vitamin D3 levels were measured in plasma samples obtained from foals from the neonate stage to the puberty stage (1 year). Two groups were compared monthly for one year: the first included foals castrated during the first week after birth and the second included non-castrated foals (Figure 5).

Puberty is characterized by an significant level of testosterone (425 ± 59.2 pg/mL) as observed for the control group at 12 months compared to castrated horses at the neonate stage (Figure 6). A low testosterone level confirmed the castration (11,93 ± 0,97 pg/mL). A monitoring of vitamin D levels was performed from blood samples collected monthly from birth to 12 months (Figure 5). For geldings, the 1α,25 (OH)2 vitamin D3 level did not present any significant variations during the first year of life (mean level: 215,05 ± 30,35 pg/mL). Noncastrated horses exhibited the same pattern with a constant level during the first year of life (mean level: 170,21 ± 17,34 pg/mL). For each sample, the two groups were compared, but the geldings did not show significantly different levels in non-castrated horses. Both groups had similar 1α,25(OH)2 vitamin D3 levels regardless of gonadal status.

1α,25(OH)2 vitamin D3 levels in stallions and horses castrated after puberty

The same analysis was carried out on horses castrated after puberty or in adulthood. A monitoring of vitamin D levels was performed from blood samples collected before castration and 3-and 6-months after castration.

A group of animals was studied at the post-pubertal stage in which castration was carried out after puberty between 18 months and 2 years (mean age: 2.2 years old). No significant difference in the vitamin D levels was observed before castration and 3 and 6 months after castration (mean level: 285,91 ± 16,03 pg/mL) (Figure 7A).

Finally, the same analysis was carried out on the last group of the study in which horses were castrated in adulthood (mean age: 6.6 years old). Likewise, the level of 1α,25 (OH)2 vitamin D3 showed no variation between stallions and geldings (mean level: 213,73 ± 15,66 pg/mL) (Figure 7B). In the absence of testis, the same circulating levels of vitamin D were observed, whether the animals were castrated or not.

Discussion

The aim of the study was to determine the putative testicular contribution to vitamin D bioactivation in equine species, as demonstrated in different species. Subsequently, the consequences of castration on circulating vitamin D levels were studied. A surprising finding was that castration did not affect circulating 1α,25 (OH)2 vitamin D3 levels. This has been observed at any stage of testicular development. An age-dependent difference has been observed in plasma vitamin D levels. To our knowledge, few studies have described the effect of age on vitamin D levels in equine species. Pozza and colleagues also described age-related differences with higher levels in adults than in yearlings, and showed no significant difference between yearlings and adults as we observed for post-pubertal and adult horses in our study [START_REF] Pozza | Serum vitamin D, calcium, and phosphorus concentrations in ponies, horses and foals from the United States and Thailand[END_REF]. However, vitamin D levels in adult horses were lower than those observed in humans. All these results suggested an absence of equine testis's contribution to the vitamin D bioactivation whatever the stage of testicular development. The gene expression study of enzymes metabolizing vitamin D confirmed this hypothesis with an absence of the expression of CYP2R1 and CYP27B1 in opposite to situation described in other species and low levels of CYP27A1 in horses's testis.

Given the studies conducted in other species, it might have been feared that castration would have an osteoarticular impact as has been demonstrated in humans or rodents. Indeed, in human, gonadal deficiency was often correlated to low level of vitamin D in plasma [START_REF] Foresta | Altered bone status in unilateral testicular cancer survivors: Role of CYP2R1 and its luteinizing hormone-dependency[END_REF]. Vitamin D deficiency is implicated in a wide range of skeletal diseases such as altered bone mineral density (BMD), bone disorders, bone mineral deficiency and increased bone fractures. However, this study did not show any avitaminosis in equine species after castration.

The specificity of equine species can be explained by its evolutionary difference compared to other species [START_REF] Uhl | The pathology of vitamin D deficiency in domesticated animals: An evolutionary and comparative overview[END_REF]. Indeed, as mentioned previously, horses have an atypical vitamin D metabolism. Vitamin D may play a less important physiological role in this species and therefore fewer organs will contribute to its bioactivation. Rickets are rare in equine species because they have developed calcium homeostasis differently in other animals. Compared to humans and rodents, calcium homeostasis is a specific characteristic of equine species with a high capacity of intestinal calcium absorption and high renal excretion [START_REF] Breidenbach | Peculiarities of vitamin D and of the calcium and phosphate homeostatic system in horses[END_REF][START_REF] Mäenpää | Serum Profiles of Vitamins A, E and D in Mares and Foals During Different Seasons[END_REF][START_REF] Schryver | The Site of Calcium Absorption in the Horse[END_REF][START_REF] Toribio | DISORDERS OF THE ENDOCRINE SYSTEM[END_REF][START_REF] Toribio | Comparison of serum parathyroid hormone and ionized calcium and magnesium concentrations and fractional urinary clearance of calcium and phosphorus in healthy horses and horses with enterocolitis[END_REF]. The renal activity of 1α-hydroxylase CYP27B1 is low, and thus results in low vitamin D levels in this species [START_REF] Toribio | DISORDERS OF THE ENDOCRINE SYSTEM[END_REF]. Therefore, CYP27B1 activity and consequently the active vitamin D metabolism depend on the calcium status. In horses, 70% of the absorbed calcium comes from their diet [START_REF] Schryver | The Site of Calcium Absorption in the Horse[END_REF]. To balance the high intestinal absorption of calcium, kidneys have become accustomed to removing large amounts of calcium. The urinary excretion of calcium in horses is 5 to 10-fold higher than in other animals or in humans [START_REF] Toribio | Comparison of serum parathyroid hormone and ionized calcium and magnesium concentrations and fractional urinary clearance of calcium and phosphorus in healthy horses and horses with enterocolitis[END_REF]. In fact, 98 to 99% of the filtered calcium is reabsorbed by the renal tubules in domestic animals [START_REF] Blaine | Renal Control of Calcium, Phosphate, and Magnesium Homeostasis[END_REF]. Vitamin D exerts its endocrine activity on calcium transporters to maintain calcium homeostasis and the low vitamin D amounts in horses suggest that paracellular calcium transport may be more important than transcellular transport and therefore less dependent on vitamin D [START_REF] Alexander | Paracellular calcium transport across renal and intestinal epithelia 92[END_REF]. Vitamin D may not play a central role in calcium homeostasis regulation in horses as has been observed in other species. Thus, in case there should be a vitamin D independence, it is likely that less tissues are involved in vitamin D metabolism. This was shown in this study with an absence of contribution from the equine testis to this bioactivation. The lack of effect may be due to the timing of the analysis. It would be interesting to study the acute effect, with analyses performed on the days following castration.

Moreover, this particularity can be linked to its herbivore status. Indeed, in evolutionary terms, calcium metabolism is more or less dependent on vitamin D. In carnivorous animals such as cats and dogs, 7dehydrocholesterol levels are low. Indeed, during evolution, these animals became more dependent on their carnivorous diet which is a good source of vitamin D and have lost the capacity to produce sufficient levels of vitamin D by skin synthesis [START_REF] How | Dietary vitamin D dependence of cat and dog due to indequate cutaneaous synthesis of vitamin D[END_REF]. On the contrary, herbivores are more dependent on endogenous synthesis. Besides, there is a vegetable form of vitamin D: Vitamin D2 (ergocalciferol). Ergocalciferol is found in fungi, yeast, grass and hay and can be a second source of vitamin, especially in equine species. However, in a grazing ruminant such as ram, almost all vitamin D comes from dermal photosynthesis and the expression of vitamin D metabolizing enzymes has been demonstrated in the male reproductive tract [START_REF] Yao | Age-associated expression of vitamin D receptor and vitamin D-metabolizing enzymes in the male reproductive tract and sperm of Hu sheep[END_REF], which makes the equine species a special model.

Conclusion

This study has highlighted the non-contribution of the equine testis in vitamin D bioactivation.

Consequently, from a practical point of view, castration did not impact long-time vitamin D levels. There is currently no specific recommendation on castration, i.e., it can be performed at any age. Regarding the vitamin D metabolism, this study did not show any contraindication to horses' castration regardless of the age at which it is performed. As equine testis did not contribute to vitamin D bioactivation, horses are an exception when compared to the other species. The statistical analyses were performed over a period of 12 months in each group using one-Way ANOVA followed by a Tukey test. The samples of both groups were compared using a t-test.

  Dulbecco's Phosphate Buffered Saline (PBS) was purchased from Pan Biotech (D. Dutscher, Aidenbach, Germany). A RT-PCR kit was purchased from Promega (Madison, USA): deoxyribose nucleotide triphosphate (dNTP), Random hexamers, Ribonuclease Inhibitor (RNasin), Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT), GoTaq PCR Master Mix were purchased from Promega (Madison, USA). Primers were purchased from Eurogentec (Seraing, Belgium). A testosterone ELISA Kit was purchased from Enzo Life Sciences (Farmingdale, USA) and 1,25 (OH)2 vitamin D3 ELISA Kit from MyBioSource (California, USA).

Figure 2 :

 2 Figure 2: Testicular expression of CYP27A1, CYP27B1 and CYP2R1 relative to ages. Identification of CYP27A1, CYP27B1 and CYP2R1 transcripts in testis from adults (lane 1 to 5), post-pubertal horses (lane 6 to 10) and neonates (lane 11 to 14) by RT-PCR. The last lane represents negative control. The PCR products were resolved on 1,5% agarose gel electrophoresis visualized by ethidium bromide staining. Molecular standard size (M) is a 100 pb DNA ladder (Biolabs). * indicates 200pb. GAPDH was used as an internal control.

Figure 3 :

 3 Figure 3: Immunodetection of CYP27A1 protein in adult equine testis. The positive CYP27A1 signal is visible in interstitial tissue. Insert: no positive staining was observed when the primary antibody was omitted (100μm scale bar).

Figure 4 :

 4 Figure 4: Circulating levels of 1α,25-dihydroxyvitamin D3 relative to the age. 1α,25-dihydroxyvitamin D3 levels were measured from neonates (n=8; 1week), post-pubertal (n=8; 2.2 years old) and adult (n=6; 6.6 years old) groups before castration. Data are shown as mean ± SEM. Statistical analyses were conducted using one-Way ANOVA followed by Tukey test (a ≠ b; p ≤ 0.001).

Figure 5 :

 5 Figure5: Circulating rates of 1α,25-dihydroxyvitamin D3 in plasma samples of foals. 1α,25dihydroxyvitamin D3 levels were measured before and after castration of foals castrated following birth and of non-castrated foals for 12 months. Data are shown as mean ± SEM (n=8). The statistical analyses were performed over a period of 12 months in each group using one-Way ANOVA followed by a Tukey test. The samples of both groups were compared using a t-test.

Figure 6 :

 6 Figure 6: Puberty in the NC group. The testosterone levels were measured in castrated and noncastrated groups at puberty stage (12 months). Data are shown as mean ± SEM. Statistical analyses were conducted using an unpaired T-test. *** p ≤ 0.001) (non-castrated n=8; castrated n=8).

Figure 7 :

 7 Figure7: Circulating rates of 1α,25-dihydroxyvitamin D3 in plasma samples of post-pubertal (A) and adult horses (B). 1α,25-dihydroxyvitamin D3 levels were measured in adults (n=6; 6.6 years old) and post-pubertal horses (n=8; 2.2 years) at 3 and 6 months after castration. Data are shown as mean ± SEM. The statistical analyses were conducted using one-Way ANOVA followed by a Tukey test.

  Figure 1

Figure 1: experimental design of collected blood samples from experimental herds.

  Blood samples were collected monthly until puberty (17 months of age) for the two experimental herds from INRAE: castrated foals following birth (n=8) and uncastrated foals (n=8). Blood samples from Saint-Michel de Livet veterinary clinic were obtained at 3 and 6 months after castration: adult (n=6; 6.6 years old) and post-pubertal horses (n=8; 2.2 years old). A blood sample was collected for all groups before castration (P0).

Acknowledgements

We thank everyone who helped us on this project. We also thank the "Carré International" of the University of Caen Normandie (France) for editing the article.

Sources of funding

This study was funded by the « Région Normandie », « Institut Français du Cheval et de l'Equitation » (IFCE) and by the « Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation » (Ministry of higher education and research).

Conflict of interests

Conflict of interests: none


Mohammed Saïb
email: mohamed.saib@cea.fr

Maxime Besacier

Christophe Constancias

P Michallon

effects in EUV interferometer lithography

Keywords: EUV Lithography, interferometer, diffraction, Fresnel diffraction, modelling, contrast

INTRODUCTION

In the microelectronics industry, Moore's Law predicts that the number of transistors in an integrated circuit (IC) doubles every 18 months. This increase leads to a decrease of their dimensions. A continuous improvement of technical and industrial equipments is mandatory to follow this law. The lithography domain, a key step in the manufacturing cycle, is concerned by this trend. The optical transmission lithography shows its physical limits to achieve, in the near future, the dimensions specified. New technologies emerge but are not still well adapted to the industrial production constraints [1,2]. Among the promising technologies, the Extreme UltraViolet (EUV) Lithography is regularly cited [3,4]. Very close to the techniques used up to now, its very short wavelength (13.5 nm) can reach nanoscale dimensions. Nevertheless, many technological barriers must be overcame to meet the requirements. In this context, the parallel development of tools should allow to characterize and analyze the issues. The interferometer appears as a technique to define geometric high resolution with a short exposure time [5][6][7]. At the EUV wavelength, an Interferometer Lithography (IL) allows characterizing the resists such as their sensitivity [8]. This article addresses, in a global approach, disturbances affecting the quality of the printed interferences. This work starts by the photometry study applied to EUV source. By considering the analytical equation of the contrast of interference fringes, some approaches to ensure a good quality of the resist patterns are proposed. These approaches must take into account the tolerated photons path difference and the optical intensity at the diffraction grating level. In this work, two main optical configurations of the interferometer are considered. For each case, the useful optical intensity is estimated. Then, the impact of the dimensions of diffraction mask on the interferences is shown. Disturbances such as Fresnel diffraction or defocusing are presented and their contribution on the interference damage is shown.

PRINCIPLES OF THE INTERFEROMETRY LITHOGRAPHY

The single grating EUV-IL experimental setup is shown in figure 1. It is composed by a light source, ideally generating a collimated beam and with a high average density of radiation. The EUV light is applied to a diffraction grating. The surface of this diffraction mask has two grating windows of width Cp and separated by an absorber layer of width d. Both windows contain a dense line grating of period p to diffract the incident light. The whole grating structure is placed on a silicon membrane of 100nm thickness. The EUV light projected onto the transparent grating windows is diffracted according to several discrete orders. The angles of diffraction orders vary depending on the light wavelength λ and the period of diffraction grating p. For a given diffracted order m, the diffraction angle θ m, defined regarding the axis of incidence of light, is written as:

p m m λ θ = sin (1)
For the 1st order diffraction, the intersection of the +1st order with the -1st order from both different gratings is responsible for the appearance of a stationary sine wave. It prints in the photoresist, a dense line grating with a period two times lower than the grating period. Since the intersection of the +1st and -1st diffracted orders occurs with an angle 2.θ m , the width of the recombination area varies with the distance between the grating mask and the resist (figure 1). The maximum width of this area is obtained for the focal distance:

⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ + = p d Cp W D λ arcsin tan 2 (2)
We introduce the Depth of Focus (DoF) as the distance between the nearest and farthest plane compared to the focal plane (W D ) for which interference fringes are printed. This is expressed:

⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ = p Cp DoF λ arcsin tan (3)

OPTICAL CONFIGURATION OF THE INTERFEROMETER

Considering an EUV beam generated by an extended source (figure 2), the circular radiation source surface with a radius s generates a divergent beam characterized by its half-divergence angle θ s . This light beam is diffracted by the grating mask and the recombination fo the +/-1 diffraction orders gives interference fringes. The resulting intensity according to the x-axis is written as:

⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⋅ + ⋅ = p x C I x I π 4 cos 1 2 ) ( 0 (4)
where x is the position of the optical intensity on the resist plane and C is the contrast of interferences fringes linked to the properties of the light beam. It is expressed as:

( ) ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ ⋅ ⋅ + ⋅ = λ δθ π 2 sin Cp d c C ( 5
)
where sinc is the sinus cardinal, δθ is the half angular width of the source observed from the grating plane. It is written as:

( ) D s tg = δθ (6)
s is the radius of the source and D the distance between the source and the grating mask plane as shown in figure 2. An arbitrary minimum value of contrast established at 63% guaranteed good quality of printed interference fringes on the resist. By putting this value in equation 5, we define the maximum source angular width admitted:

( )

Cp d + ⋅ ≤ 4
λ δθ (7) Two possible configurations are considered in following subsections to respect the condition of the equation 7. The source is defined by it angular width δθ, and delivers a beam characterized by it half angle divergence θ s .

Distance EUV source -diffraction grating

The first approach is to improve the partial collimation of the beam by the setting of the distance between the EUV source and the diffraction grating. If the source is far enough to the grating, the beam rays are considered nearly parallel.

It means if the distance D is () δθ tg s D =
. On the other hand, the EUV source must provide enough energy at the desired wavelength to carry out prints in a reasonable time. The optical intensity per grating window surface is written:

( ) ( ) 2 2 s 2 1 s tg D L Cp F Sg Sw F I + θ ⋅ π ⋅ ⋅ = ⋅ = ( 8
)
where F is the flux of the EUV source, Sw the surface of each grating window, Sg the surface of the EUV beam at the level of the grating plane, D the distance between the source and the diffraction grating, and L the length of the grating window.

The following example gives some numerical values: Considering a diffraction grating with d = Cp = 30μm, and length L=800µm, irradiated with light wavelength 13.5nm by an EUV source with a radius of 400μm. This source delivers a photon flux 10 16 ph/s at the level of its output. It has a half divergence angle of 5.58mrad. These dimensions leads to a half angular width δθ ≤ 56.3µrad as expressed in the equation 7. It also induces a distance D = 704cm. For this distance D, the optical intensity I 1 is 9.8 10 8 ph/(s⋅Sw). Note that the distance of the source at 704cm from the diffraction grating is relatively difficult to implement for experiments.

The diaphragm insertion

In the second optical configuration, the improvement of the partial light collimation is made by the cancellation of photons which not ensure the condition described by the equation 7. This is done by spatially filtering the light source. It consists of a diaphragm insertion with a radius R on the EUV beam (figure 3). The radius of the diaphragm is determined depending on the distance D 2 :

() δθ tg D R ⋅ = 2
(9) This distance D 2 must be as low as possible to have the maximum intensity on gratings. In order to reduce the photon loss, the diaphragm is placed at the source exit. Since the y-axis does not require any filtering, the orders recombination does not occur in this direction, the circular diaphragm can be replaced by a rectangular slit of width 2R and length L equal to the length of transparent windows grating. In this case, the optical intensity per grating window surface is written:

( ) (
)

) ( 2 ) ( ) ( ) ( 2 2 2 2 2 2 2 s s tg D L tg D tg D s tg D Cp L F I θ δθ θ π δθ ⋅ ⋅ + ⋅ + ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ = ( 10
)
The numerical application with the same configuration described in 3.1 and with the slit position at a distance D 2 = 20cm (arbitrarily chosen) shows that the slit shape will be rectangular with a width 22µm and a length (L) 800µm. The EUV source provides in this case an optical intensity I 2 equal to 1.27·10 12 ph/(s·Sw).

The numerical applications for both configurations show that using a slit in the interferometer set-up is interesting since it allows putting the source at a reasonable distance of the diffraction grating (20cm instead 704cm) while keeping the collimation of the source beam. The intensity I 2 is then is higher than I 1 .

EXPERIMENTAL RESULTS

In this section, experimental results of prints made at the EUV wavelength are shown and compared with modeling results. The undesirable effects on the prints induced by the diffraction mask are addressed. They are divided into three disturbances. First, the Fresnel diffraction effect which affects the profile of the interferences fringes area printed in resist. The second effect, which reduces the interference area width, is linked to the position of the resist plane out the focal plane. The third effect called shadowing effect causes also the decrease of the interference area by absorption of diffracted orders near the edges of gratings windows Note that this last effect is easy to avoid, regarding some constraints on experimental process. The print tests have been carried out thanks to the EUV synchrotron source Aladdin from SRC (Synchrotron Radiation Center) of Wisconsin [9]. In the experimental tool, the diffraction grating is put on a 3 moving axis stand to address multiple resist prints. The exposure time (close to 1second) is tuned for each experimental configuration to insure a good print quality. The diffraction mask is built on a silicon membrane and has four different couples of gratings with periods of 200nm, 140nm, 100nm and 80nm [10]. The focal distance W D remains the same for the four grating and is equal to 600µm. It means that the distance d is different and adjusted for each couple of gratings. The width of windows Cp is 50µm for 200nm and 100nm gratings and 60µm for 140nm and 80nm gratings. The total length L of gratings is 800µm. This experimental system is put within a vacuum vessel and is mechanically stabilised.

The Fresnel diffraction effect

The first effect addressed is the Fresnel Diffraction [START_REF] Born | Principles of optics[END_REF]. The effect is a boundary effect and it appears when the light passes through the grating transparent windows. This phenomenon is resulted by a non uniform and localized variation of the transmitted optical intensity. This overlapping with the fringes, in the interference area, induces a damage of local interferences shape especially if the Fresnel intensity shows some significant variations (figure 4). The profile of this intensity can be determined regarding the following parameters: the wavelength, the distance between the grating and the resist and the width of the transparent window. In the diffraction theory, the Fresnel number gives a numerical definition of this profile with the following expression. A strong intensity variation is then observed in the area of the diffraction window. The intensity ratio between center and border values is close to 350%. The W exp decrease leads to a N F increase (figure 5b). In this case the intensity profile remains inhomogeneous on the width Cp. 3 undulations are observed. For N F =9 (figure 5c), the intensity window is larger and the mean value is steady. It remains, nevertheless some important local undulations. Finally for a high N F =25 (figure 5d), the intensity shows undulations with a weak magnitude, especially for the center area ,far from the boundaries of grating. The contrast of one undulation is defined by the following relationship: min max min max

I I I I C + - = (12)
With I max and I min the extremum intensity values of the same undulation.

For instance, on the figure 5d, the contrasts of the five first undulations from the border to the center are respectively 28%, 16%, 13%, 10% and 9%. For N F > 25, the Fresnel diffraction curves keep the same profile than N F = 25, then it influence on the interference fringes remains the same. The Fresnel effect is superimposed to all diffracted orders. In the interference area, the Fresnel effect causes a disturbance of the profile of the dense lines profile printed in resist. The best way to only observe this effect is on the 0th order, it means without any other effects such as interferences fringes. The figure 6a shows a MEB picture of the printed resist at the 0th order. In this case the distance between diffraction grating and the resist plane is 560um. Printed patterns are observed within the rectangle of the diffraction window. The diameter of these patterns decreases when going to the exposed surface center. Beyond the nineth pattern, disturbances printed in the resist vanish and the surface becomes homogeneous. The nineth undulation is considered as the last undulation of disturbance. The contrast of this undulation is 7%. The Fresnel effect is also modeled as shown in figure 6b The figure 7 shows a modeling case for a Fresnel number N F < 3 leading to a strong inhomogeneous diffracted intensity on 0 th order. To obtain this picture, the dimensions configuration is the following:

Cp = 5um L = 800um W exp = 2mm
The N F is 0,23 in the x axis and 5925 in the y axis. Intensity values on boundaries (in blue color) are five times lower than those in center (in red color). The resulting contrast is close to 60%, strongly higher than the 7% acceptable. This configuration causes a degradation of the fringe contrast in the middle of the interference area. This configuration has not been experimentally implemented but it shows that a low Fresnel number on an axis is not available for a diffraction mask design. In order to avoid this kind of situations, the following condition is applied on the diffraction window width: (13) For an interferometer at 13.5nm and a diffraction grating with a working distance at 600µm, the width Cp must be larger than 29µm.

The Focus effect

The diffraction mask causes a second disturbance which is the decrease of the interferences area. Regarding the principle of the interferometer (figure 1), the interferences appear on an area width Cp which is the width of the diffraction gratings if the focal distance is respected. However, the experiments show that the interferences area can be reduced if the experimental distance between the gratings and the resist plan is different to the focal distance (600µm). The figure 8 shows a global view of the interferences area printed in the resist by a grating with a width Cp = 60µm and a working distance W exp = 560µm. Three different areas can be distinguished. A grey central area contains the printed periodic lines with a period of 70nm and along the y-axis. On top and bottom sides of this central area, two other darkest areas are observed with a width 2x p (figure 9). There is no print within. Indeed, since the resist is out of the focal distance, there is a decrease of the interference area of x p on each side compared with the optimal width at the distance focal. This decrease is expressed by the equation 14: Since there is only one order in these both areas (+1 or -1) no interferences fringes can be observed and the absence of these dark areas on lateral sides of the picture shows that it is directly dependent on the direction of the diffraction orders. For the experiment leading to the figure 8, the grating period is 140nm and the working distance is 560µm whereas the focal distance is 600µm. It leads to a width decrease of 7,6µm of the interference print area (3,8µm on each side). The width decreases from 60µm to 52,4µm.

⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⋅ - = p tg W W x D p λ arcsin exp (14)
In spite of the straight shape of the boundaries of the diffraction windows, the dark areas on top and bottom sides shows some disturbances at edges 1, 2, 3 and 4 (figure 8) along the y axis and look like Fresnel diffraction previously observed on z axis (see section 4.1). The diffraction on the first order is oblique regarding the z axis and exists only in the xoz plan (there is no diffraction in the yoz plan). +1 and -1 orders of diffraction have also Fresnel diffraction phenomenon on their propagation axis (figure 10). The projection of these Fresnel diffraction effects in the resist plan shows an inverse shape of Fresnel figures on edge 1 and edge 2 and on edge 3 and edge 4, mainly because that edges 1 and 3 are provided by the -1 diffraction order of the left grating while edges 2 and 4 are provided by the +1 order of the right grating.

The Shadowing effect

Another effect which leads to disturbances on the interference area is dependent on the position and on the height of the absorber layer between both diffraction gratings (figure 9). The incident light comes to gratings with a normal incidence to the grating plan. Close to boundaries of the diffraction window, the diffracted light ray runs up against the absorber layer. For a given absorber layer with a height h, the decrease of the width of the interference area on each side is:

⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⋅ = p h x n λ arcsin tan (15)
The total width decrease due to shadowing effect is then 2x n . Considering the order of magnitude of the height h, this effect on the width decrease remains low compared with the working distance effect (focalisation effect). For a grating with a 140nm period and an absorber height of 0,4µm, the resulting decrease is 0.048µm. It shows that this effect is less disturbing compared to the defocus effect. Moreover, this effect can be cancelled by a flip of the absorber pattern (face to the light source and not face to the resist anymore). It must be considered for the experimental exposures.

CONCLUSIONS

A fully detailed study of disturbances affecting interference fringes obtained thanks to an EUV interferometer is presented. It is based on an experimental campaign on an EUV synchrotron source and on the modeling of phenomena occuring in this kind of structures during the interference print process. Considering the contrast and the size of the interference area as key parameters, disturbance causes can be clearly identified and their impacts are estimated: The EUV beam divergence effect leading to a contrast damage is reduced thanks to put a slit in front of the light source allowing a spatial filtering and only keeping EUV photons available for a good resist print quality.

-The Fresnel effect generated by the grating diffraction leads to a decrease of interference contrast on interference area boundaries for a Fresnel number N F < 25.

-The defocusing of the resist plan on the diffraction grating plan leading to a decrease of the width of the interference area.

-The oblique incidence of the +1 and -1 diffracted orders leads to a disturbance of the boundaries of the interference area.

Figure 1 .

 1 Figure 1. Interference lithography principle using a single grating mask. The grating surface is composed by two grating windows (Cp width), separated by a distance d. The +1st order and -1st order interfere and generate fringes with half grating pitch.

Figure 2 .

 2 Figure 2. Schematic representation of source of diameter s and surface Sp positioned at distance D from interferometer grating mask.

Figure 3 .

 3 Figure 3. Schematic representation of a source spatially filtered by a slit insertion of width 2R and at a distance D 2 from grating mask.

Figure 4 .

 4 Figure 4. Schematic representation of optical intensities delivered to the resist for printing. Two intensities are shown: The interference fringes intensity which has a period of an half of the mask grating period and the Fresnel intensity which has a variable period of 4µm near boundaries. Note that this scheme does not respect the period scale. 4 examples of diffracted intensities computed for an EUV light are shown in figure 5. The comparison of these four figures shows the intensity profile evolution versus the Fresnel number. The case N F =0,75 corresponds to the most important distance W exp .A strong intensity variation is then observed in the area of the diffraction window. The intensity ratio between center and border values is close to 350%. The W exp decrease leads to a N F increase (figure5b). In this case the intensity profile remains inhomogeneous on the width Cp. 3 undulations are observed. For N F =9 (figure5c), the intensity window is larger and the mean value is steady. It remains, nevertheless some important local undulations. Finally for a high N F =25 (figure5d), the intensity shows undulations with a weak magnitude, especially for the center area ,far from the boundaries of grating. The contrast of one undulation is defined by the following relationship:

Figure 5 .

 5 Figure 5. Fresnel diffraction intensity shape for: (a) N F =0.75, (b) N F =3, (c) N F =9 and (d) N F =25. The brown rectangle represents the diffraction window width.

Figure 6 .

 6 Figure 6. a) SEM image of resist patterned under 0th order diffracted by the grating window of 800μm in length and 60μm in width. The exposure is performed at work distance W ext =560μm. b) 2D modelling of the aerial image of the diffracted intensity with 0th order. This computation is performed for the same structural and experimental dimensions with Fig. 6a.

  of these disturbances. The diffraction window width must then check the relation:

Figure 7 .

 7 Figure 7. 2D modelling of the diffracted intensity by rectangular grating window of 800µm in length, 5µm in width and focal plane at 2mm. White rectangle outlines the limits the diffraction window.

Figure 8 .

 8 Figure 8. Overview image of the interference region printed in the resist using a grating period of 140nm. Interferometric fringes are localized in the central region (bright) of rectangle. Dark regions on top and bottom side of image appear because of the gap between the focal distance (600μm) and the experimental work distance (560μm).

Figure 9 .

 9 Figure 9. Schematic illustration of single grating interferometer. Coherent light is diffracted into -1st and +1st orders and give a maximum interference width at focal distance. For work distance out of the focal plane, the width decrease gradually. The 1st order diffracted near tot the edge of grating windows is absorbed by absorber layer (shadowing effect).

Figure 10 .

 10 Figure 10. Schematic representation of +1st order light diffracted by a grating. The Fresnel diffraction effect is superimposed to the 1st order and it component according x-axis is printed.

Acknowledgment:

The authors would like to acknowledge J. Wallace of the center for nanotechnology of Wisconsin-Madison University through experimental collaboration at the synchrotron radiation center.


Fanny Roussin

Abdelkader Taibi
email: abdelkader.taibi@hotmail.fr

Mireille Canal-Raffin

Lucile Cantournet

Sylvaine Durand-Fontanier

Michel Druet-Cabanac

Souleiman El Balkhi

Gaelle Maillan

Roussin Fanny

Taibi Abdelkader

CANAL-RAFFIN Mireille

Cantournet Lucile

Durand-Fontanier Sylvaine

Druet-Cabanac Michel

EL Balkhi Souleiman

Maillan Gaëlle

Assessment of workplace environmental contamination and occupational exposure to cisplatin and doxorubicin aerosols during electrostatic pressurized intraperitoneal aerosol chemotherapy

Keywords: peritoneal metastasis; electrostatic precipitation, pressurized intraperitoneal aerosol chemotherapy, antineoplastic drugs, safe handling, occupational exposure, operating room contamination

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

Introduction

Peritoneal metastasis (PM) is a terminal illness that is treated with systemic chemotherapy and palliative care. Although the new chemotherapeutic agents and targeted therapies are more effective, their efficacy is hampered by heterogeneous drug distribution and poor tissue penetration in PM, preventing improvement in the overall survival of patients with unresectable PM [START_REF] Elias | Peritoneal colorectal carcinomatosis treated with surgery and perioperative intraperitoneal chemotherapy: retrospective analysis of 523 patients from a multicentric French study[END_REF][START_REF] Rivera | Chemotherapy of advanced gastric cancer[END_REF]. Furthermore, intraperitoneal chemotherapy is designed to maximize the chemotherapeutic dose by delivering the antineoplastic drugs directly to the PM, while minimizing systemic toxicity [START_REF] Goodman | Chemotherapy for intraperitoneal use: a review of hyperthermic intraperitoneal chemotherapy and early post-operative intraperitoneal chemotherapy[END_REF].

Pressurized intraperitoneal aerosol chemotherapy (PIPAC) has been described as a new and innovative therapeutic approach to intraperitoneal chemotherapy administration in experimental and clinical studies [START_REF] Girshally | Pressurized intraperitoneal aerosol chemotherapy (PIPAC) as a neoadjuvant therapy before cytoreductive surgery and hyperthermic intraperitoneal chemotherapy[END_REF][START_REF] Robella | Safety and feasibility of pressurized intraperitoneal aerosol chemotherapy (PIPAC) associated with systemic chemotherapy: an innovative approach to treat peritoneal carcinomatosis[END_REF][START_REF] Solass | Pressurized intraperitoneal aerosol chemotherapy (PIPAC): occupational health and safety aspects[END_REF]. This innovative concept enhances the penetration of intraperitoneal chemotherapeutic drugs in the PM by taking advantage of the physical properties of gas and pressure to overcome the pharmacological limitations [START_REF] Giger-Pabst | Comparison of Tissue and Blood Concentrations of Oxaliplatin Administrated by Different Modalities of Intraperitoneal Chemotherapy[END_REF]. Reymond et al. proposed combining nebulization of chemotherapy with electrostatic precipitation using the Ultravision TM system to decrease the time of the surgical procedure and enhance the pharmacologic effect of PIPAC by increasing the in situ bioavailability of the used drugs [START_REF] Kakchekeeva | In Vivo Feasibility of Electrostatic Precipitation as an Adjunct to Pressurized Intraperitoneal Aerosol Chemotherapy (ePIPAC)[END_REF][START_REF] Graversen | Pressurized IntraPeritoneal Aerosol Chemotherapy with one minute of electrostatic precipitation (ePIPAC) is feasible, but the histological tumor response in peritoneal metastasis is insufficient[END_REF]. More recently, some teams performed these PIPAC procedures associated to an electrostatic field in order to enhance charged droplet precipitation and tissue penetration. For electrostatic pressurized intraperitoneal aerosol chemotherapy (e-PIPAC), the electrostatic generator is activated after chemotherapy aerosolization and the exposure time after activation is 6 minutes (min), shorter than PIPACs procedures (30 min) [START_REF] Taibi | Pressurized Intraperitoneal Aerosol Chemotherapy Enhanced by Electrostatic Precipitation (ePIPAC) for Patients with Peritoneal Metastases[END_REF]. Nevertheless this electrotherapy requires the installation of a third trocar without "balloon protection", for introduce an atraumatic stainless steel brush electrode. This trocar may increase the risk of contamination in the operative room (OR) during ePIPAC and thus, the hypothesis of study was that ePIPAC increased the risk of contamination.

Because of their teratogenic and mutagenic effects, cisplatin and doxorubicin [START_REF] Connor | Reproductive health risks associated with occupational exposures to antineoplastic drugs in health care settings: a review of the evidence[END_REF], which are regularly used for PIPAC and electrostatic PIPAC (ePIPAC) [START_REF] Alyami | Pressurised intraperitoneal aerosol chemotherapy: rationale, evidence, and potential indications[END_REF], are classified as probably carcinogenic to humans (group 2A) by the International Agency for Research on Cancer [START_REF]Monographs on the evaluation of carcinogenic risks to humans[END_REF].

The health risk is influenced by the level and frequency of exposure, the toxicity of the drug, the existence of proper work practices and among other factors. In order to help medical personnel, european agency for safety and health at work proposes health-based indicative some values to evaluate this risk (occupational exposure limit values (IOELVs), occupational exposure limits (OELs) -binding limit values (BOELVs)). ( 14) Thus, this innovative concept of aerosol administration of cisplatin and doxorubicin has raised safety concerns regarding the exposure of medical and paramedical teams. Nevertheless, some studies have evaluated the cisplatin exposure via operating room (OR) air and workplace surfaces, determined by air and wipe sampling, and PIPAC staff gloves. The rates of contaminations were low but variables between the studies, the areas evaluated of OR and depending on the analysis technique used. Ndaw et al showed that the median concentration in the PIPAC group is < LQ of 10 ng/L and a maximum concentration of 136 ng/L were measured [START_REF] Ndaw | Occupational exposure to platinum drugs during intraperitoneal chemotherapy. Biomonitoring and surface contamination[END_REF]. For e-PIPAC, only one study was published and these authors concluded that it had not any surface, air, or material contamination with platinum during or after two e-PIPAC procedures [START_REF] Willaert | Occupational safety of pressurized intraperitoneal aerosol chemotherapy (PIPAC)[END_REF]. However, no study has evaluated exposure to doxorubicin during ePIPAC.

Here, we report toxicological analysis (Cisplatin and/or Doxorubicin) including air samples, surface wipe samples, and analysis of gloves and urine samples after two e-PIPAC procedures.

Methods

Each ePIPAC procedure was performed after validation by an interdisciplinary meeting of members in our expert centre. All patients had unresectable PM without a curative treatment option and were candidates to receive systemic chemotherapy associated with ePIPAC (cisplatin with doxorubicin).

The study was conducted on two consecutive ePIPACs (A and B), performed by the same surgical team in the digestive surgery operating room (OR). Occupational exposure to cisplatin and doxorubicin was investigated by analysing environmental (workplace surfaces) and biological (urine) samples obtained from the surgical team (surgeons, anaesthetist, nurses and auxiliary nurse who did the cleaning) and a non-exposed control group (two pharmacy residents). The air samples were collected in order to detect Cisplatin contamination.

ePIPAC procedure

The ePIPAC procedure was performed under general anaesthesia. After open laparoscopy and insufflation of 12 mm Hg pneumoperitoneum, two balloon safety trocars and an active cable terminating in an atraumatic stainless steel brush electrode were inserted into the abdominal wall. Evaluation of the peritoneal cancer index was performed systemically, and biopsied specimens were obtained from each abdominal quadrant, if possible, and analysed to evaluate the peritoneal regression grading score. Nebulization was performed using a high-pressure injector inserted into the peritoneal cavity at 0.5 mL/s and 20 bars to release the chemotherapeutic agents as an aerosol into the abdomen. Before or at completion of aerosol administration, a generator unit (Ultravision system, voltage 7500-9500 V, current ≤10 µA) was activated to produce electrostatic attraction of the negatively charged aerosol particles to the PM and the peritoneal tissue surfaces in the contained space, during 6 minutes. A closed system with zero flow was maintained during this procedure at 37°C. Antineoplastic drugs aerosols were exhausted over a closed waste system (PlumeSafe® Turbo).

Floor in the operating room were cleaned using a detergent disinfectant solution (Surfanios premium®, Anios) and surfaces with a detergent solution (Polyactif, Rochex) by the operating room cleaners. Briefly, cloths were moistened with the detergent disinfectant solution and surfaces were wiped with the cloths.

Environmental measurements

Air contamination i) Analysis of environmental air samples for the presence of platinum

The particle measurements were performed by an accredited independent organization (APAVE, France). Aerosol sampling was performed using closed-face filter cassettes provided by APAVE. The airstream was filtered using a PTFE filter and a sampling pump operated at 2 L/min, GilAir®. We used stationary and personal inhalable aerosol sampling.

Firstly, cassettes were placed near the filtered return-air grille and on the PIPAC injector, at a distance of 1 m from the operating table. Personal sampling method permitted the characterization of the concentration of inhalable-sized particles within the breathing zone of an individual. Therefore, the third cassette was attached to the surgeon's glasses, pointing toward his mouth. Air sampling was initiated after inserting the trocar into the patient's abdomen and was terminated after closure (Figures 1,2).

Surface contamination (one sample by area) i) Wipe samples from surfaces

Surface samples were collected after the ePIPAC procedure and after the cleaning procedure (Figure 3). Wipe sampling was performed using pre-impregnated wipes (Ghost Wipes TM , Environmental Express®, 15 × 15 cm) to analyse platinum levels, according to a French personnel safety network (Institut National de Recherche et de Sécurité). The wipe sampling (5 × 5 cm, Medicomp® compress) procedure performed to analyse doxorubicin levels involved using a surface metrology kit developed by the Pharmacology and Toxicology Laboratory of Bordeaux University Hospital [START_REF] Willaert | Occupational safety of pressurized intraperitoneal aerosol chemotherapy (PIPAC)[END_REF]. Samples were collected from various surfaces, including several areas on the floor (10 × 10 cm), the injector screen, the cover sheet and the doxorubicin/cisplatin perfusion bag. To avoid cross-contamination, the same operator performed all wipe samplings, and each wipe was held using single-use sterile disposable tweezers.

ii) Wipe samples from gloves and overshoes

Overshoes and hands were sampled by wiping the palms, dorsal areas and interdigital spaces to determine the doxorubicin concentrations. The overshoes and gloves were placed in 180mL polypropylene containers recovered with 50 mL of 1% nitric acid and agitated for 1 hour for desorption of platinum. Doxorubicin and platinum concentrations were expressed in pg/wipe with a wipe seize of 5 × 5 cm and in ng/wipe with a wipe seize of 15 × 15 cm, respectively or pg/cm 2 and ng/cm 2 respectively.

Safety protocol

Description

All surgeons and OR personnel underwent ePIPAC training before implementation of the ePIPAC procedure. The ePIPAC procedures were performed in an OR without laminar airflow but with use of personal protective equipment and safety measures. As recommended, the OR staffs used disposable impervious gowns, FFP3 facemasks, protective glasses, double latex gloves and overshoes. Absorbent mats were positioned on the floor under the operating table and injector. The patient and all connections were secured by an additional plastic cover and smoke evacuation device (PlumeSafe® Turbo-Filtration). Nobody remained in the operating room during the ePIPAC procedure.

Efficacy: biological measurements i) Urine samples collection

Each ePIPAC staff participant collected three urine samples: the first before the ePIPAC procedure (at 7:00 AM, [day 0]), the second 6 hours after the ePIPAC procedure (at 18:00 PM; [day 0]) and the third in the next morning at wake up (7:00 AM, [day 1]). The urine samples (V=20ml) were collected in a polypropylene bottle and were then stored immediately at +4°C for the platinum until analysis and at -20°C for the doxorubicin until analysis. Urine samples were also obtained from the control group. All participants answered a questionnaire regarding their occupational and personal exposures to platinum (history of contact with chemotherapeutic drugs or medical devices such as breast or dental prostheses) and doxorubicin.

Analytical methods

Cisplatin was determined by the analysis of platinum using inductively coupled plasma-mass spectrometry. Doxorubicin was analysed by ultra-high-performance liquid chromatographytandem mass spectrometry as described by Villa et al [START_REF] Villa | A highly sensitive UHPLC-MS/MS method for urine biological monitoring of occupational exposure to anthracycline antineoplastic drugs and routine application[END_REF]. The limit of detection (LOD) of cisplatin was 10 ng/wipe for the wipe samples, 5 ng/filter for the air samples. The limit of quantification (LOQ) of cisplatin was 25ng/wipe for the wipe samples, 12.5ng/filter for the air samples and 2.5ng/L for the urine samples. The limit of detection (LOD) of doxorubicin was 5ng/L for the urine samples. The LOQ of doxorubicin was 5pg/wipe for the wipe samples and 10ng/L for the urine samples. A sample was considered positive for the antineoplastic drug when a drug concentration exceeded the LOQ value or when the drug was detected at trace level (concentration between the LOD and LOQ value of the analytical method).

Patient data

Age, sex, systemic chemotherapy, ePIPAC procedure (drug, time of chemotherapy nebulisation and time of electrostatic application) and the delay between ePIPAC and systemic chemotherapy were recorded. Adverse events were classified according to the Common Terminology Criteria for Adverse Events, version 5.0.

All procedures were performed according to the principles of the Declaration of Helsinki. All patients and surgical teams were extensively informed of the study protocols and agreed to participate in this study. None analysis was performed on the patients, no ethics committee approval was required for this study.

The first ePIPAC procedure was performed in a 64-year-old male with PM derived from stomach adenocarcinoma. ePIPAC of 13.8 mg (7.5 mg/m 2 ) cisplatin and 2.76 mg (1.5 mg/m 2 ) doxorubicin was performed, concomitant with systemic chemotherapy (folinic acid, 5fluorouracil and oxaliplatin). The second procedure was performed the same day in a 73-year old female patient with PM derived from ovarian cancer. ePIPAC was initiated using 10.4 mg (7.5 mg/m 2 ) cisplatin and 2.07 mg (1.5 mg/m 2 ) doxorubicin concomitant with systemic chemotherapy (gemcitabine). ePIPAC was performed for 12 min (6 min of intraperitoneal chemotherapy electroporation and 6 min of electrostatic therapy). We used a 2 mm surgical trocar in the first case and a 5 mm surgical trocar in the other case for electrostatic therapy.

Statistics

Only descriptive statistics are provided. Qualitative variables are expressed as percentages and quantitative variables as means. Statistical analyses were performed using Microsoft Excel 2011 (Microsoft Inc., Redmond, WA, USA).

Results

Environmental measurements

Air contamination

Six atmospheric samples were collected during the two ePIPAC procedures. Cisplatin was not detected in any of the air samples (<5 ng/filter).

Surface contamination 1.1 Floor samples

Two different analytical methods were used to analyse wipe sample involving two different type of wipe. A total of 152 samples were collected during each ePIPAC procedure and 152 * 2 assays were performed (n= 152 for cisplatin and n = 152 for doxorubicin). i) Doxorubicin: the greatest amount of doxorubicin was found on the floor around the operating table on the left side (7.2 pg/wipe = 0.29 pg/cm 2 ). Four floor samples remained contaminated after cleaning. Three samples had greater contamination after cleaning than after the ePIPAC A procedure (Figure 4 and Annex 1).

ii) Cisplatin: the greatest amount of platinum was found on the floor under the PIPAC injector (215,930 ng/wipe = 959 ng/cm 2 ). All samples remained contaminated after cleaning, with concentrations up to 32 times higher (50 ng/wipe (0.22 ng/cm 2 ) after the ePIPAC B procedure and 1 580 ng/wipe (7 ng/cm 2 ) after cleaning. Except for the most contaminated point (215,930 ng/wipe = 959 ng/cm 2 ) after the ePIPAC B procedure, in which cleaning seems to reduce contamination, all other analyses revealed higher quantities after cleaning (Figure 5 and Annex 2).

Other surface samples immediately after ePIPAC

The concentrations measured on other work surfaces after both ePIPAC procedures ranged from traces (below the LOQ) to 871.8 pg/wipe (34 pg/cm 2 ) for doxorubicin and from traces to 34,460 ng/wipe (153 ng/cm 2 ) for cisplatin. The highest concentrations of both drugs were found on the nebulizer nozzle after procedure completion and after removing the nozzle from the patient's abdomen (control analysis). Another sample from the PIPAC injector screen showed significant doxorubicin and cisplatin contamination, with concentrations of 84.9 pg/wipe (3.4 pg/cm 2 ) and 270 ng/wipe (1.2 ng/cm 2 ), respectively, for the ePIPAC A and 128.2 pg/wipe (5.13 pg/cm 2 ) and 220 ng/wipe (0.98 ng/cm 2 ), respectively, for ePIPAC B. The perfusion bag was contaminated after both procedures ePIPAC; the cisplatin concentrations were 60 and 20 ng/wipe (0.27 ng/cm 2 and 0.09 ng/cm 2 ). While only trace amounts of doxorubicin were found after the ePIPAC B procedure. (Table 2).

Gloves and overshoes samples

All of the samples were contaminated with cisplatin, and approximately half were contaminated with doxorubicin. The nurse had the two most contaminated samples corresponded to the step after fixing the perfusion bag into the injector and connecting the line (doxorubicin, 1,121 pg/wipe [44.8 pg/cm 2 ]) and after cleaning the PIPAC injector following the procedure (cisplatin, 42,100 ng/device [292.4 ng/cm 2 ]). Both of the surgeon's gloves were always contaminated with each drug. All overshoes were contaminated with cisplatin, with higher contamination after the ePIPAC B than ePIPAC A procedure.

Biological measurements

Urine samples

Doxorubicin and cisplatin were non detectable (<5 and 2.5 ng/L, respectively) in all 22 urine samples obtained (exposed and control groups).

Discussion

PIPAC may expose OR personnel to antineoplastic drugs. Occupational safety precautions have been considered since PIPAC was first introduced. Consequently, measures to minimize surface contamination in the OR and exposure of the OR personnel were documented by Reymond et al. [START_REF] Solass | Pressurized intraperitoneal aerosol chemotherapy (PIPAC): occupational health and safety aspects[END_REF]. Nevertheless, a new PIPAC procedure was described using electrostatic therapy. This technique is slightly different from standard PIPAC because it uses a third trocar without a balloon unlike other trocars (8-10). Thus, few data are available on the levels of environmental contamination and occupational exposure during ePIPAC procedures. Few data have been published on the levels of environmental contamination and internal contamination of OR personnel during PIPAC, and results are sometimes difficult to compare with others' findings due to the use of different methodologies among hospital centres and/or different analytical methods with different level of sensitivity (LOQ). In the present study, for the first time, exposure of healthcare professionals to doxorubicin following ePIPAC has been assessed. The doxorubicin analytical method [START_REF] Villa | A highly sensitive UHPLC-MS/MS method for urine biological monitoring of occupational exposure to anthracycline antineoplastic drugs and routine application[END_REF] used for this study has a low LOQ compared with most methods reported in the literature (high sensitivity). The wipe sampling method used was reproducible because the samples were all collected by the same person, with a validated sampling procedure. Air samples, which are generally difficult to implement, were collected by an accredited company, certified by the French organism of accreditation (COFRAC), thus guaranteeing the reliability of the equipment used.

or trocars, including disconnecting the high-pressure lines, primary leakage, spillage during ventilation, and connection, disconnection or removal of the syringe could be encountered.

Cisplatin and doxorubicin were detected on all surface types, with maximum concentrations of 959 ng/cm 2 and 0.29 pg/cm 2 , respectively. After cleaning the floor according to a standardized protocol, residual cisplatin and doxorubicin contamination was detected (near the absorbent mats), with maximum concentrations of 9.5 ng/cm 2 and 0.27 pg/cm 2 , respectively. Despite cleaning between the two PIPACs, cross-contamination occurred due to inefficient cleaning. The efficacy of chemical decontamination of chemotherapy work surfaces depends on the cleaning solution used, as well as the cleaning protocol. According to the literature, detergent disinfectant based on quaternary ammonium or surfactants are effective in reducing the contamination of floor contaminated with an antineoplastic drug [START_REF] Queruau Lamerie | Evaluation of decontamination efficacy of cleaning solutions on stainless steel and glass surfaces contaminated by 10 antineoplastic agents[END_REF][START_REF] Adé | Comparison of Decontamination Efficacy of Cleaning Solutions on a Biological Safety Cabinet Workbench Contaminated by Cyclophosphamide[END_REF]. This suggests that the detergent disinfectant product used in our study was suitable, but the cleaning procedures must be revised to incorporate multiple single-use mops for the floor cleaning. Thus, we found that overall contamination was higher in ePIPAC B than in ePIPAC A. This cumulative effect can be explained because the second mop used after ePIPAC B was saturated with "detergent-disinfectant product and antineoplastic drug", and have spread the contamination in many part of OR. The use of separate mops in each part OR (1 mop by 10-15m2) to prevent spreading contamination is, hence, shown mandatory. Every step of the cleaning procedure should be tested for cleaning efficiency in futures studies.

Moreover, the PIPAC injector screen and nebulizer nozzle were both contaminated, as reported in other studies [START_REF] Ndaw | Occupational exposure to platinum drugs during intraperitoneal chemotherapy. Biomonitoring and surface contamination[END_REF][START_REF] Ametsbichler | Occupational exposure to cisplatin/oxaliplatin during Pressurized Intraperitoneal Aerosol Chemotherapy (PIPAC)?[END_REF]. Contamination of a reusable device can result in staff exposure. The injectors are often stored in the anteroom of the OR, where staff eventually touch the surfaces without gloves and can be potentially exposed. We recommend to protect PIPAC injector with a single-use transparent film, as suggested by Ametsbichler et al [START_REF] Ametsbichler | Occupational exposure to cisplatin/oxaliplatin during Pressurized Intraperitoneal Aerosol Chemotherapy (PIPAC)?[END_REF].

In a study on occupational safety during ePIPAC, Willaert et al. did not detect platinum contamination on either the inner or outer pair of gloves of the surgeon ( 16). Yet, in our study, all gloves and overshoes of the ePIPAC staff were contaminated with cisplatin and some with doxorubicin, probably due to the highly sensitive analytical method (200 times higher than

Willaert et al.).

Contamination of the surgeon's outer gloves is expected due to removal of trocars and the laparoscopic incision closure [START_REF] Ndaw | Occupational exposure to platinum drugs during intraperitoneal chemotherapy. Biomonitoring and surface contamination[END_REF]. Ametsbichler et al. reported platinum contamination on the surgeon's gloves after fixing and removing the drug syringe, cleaning the PIPAC injector and closing the patient's abdomen [START_REF] Ametsbichler | Occupational exposure to cisplatin/oxaliplatin during Pressurized Intraperitoneal Aerosol Chemotherapy (PIPAC)?[END_REF]. In our case, the nurse's gloves were the most contaminated. These results are consistent because the nurse handles the perfusion bags and PIPAC injector, both of which were contaminated with the two drugs. In addition, the perfusion bag could be contaminated during fabrication before the ePIPAC procedure. This observation has already been made in the study by Ndaw et al. (15) on the evaluation of occupational exposure to cytotoxic drugs carried out in 2 French hospitals, since their surface samples in oncology departments showed that the most contaminated surfaces were in particular the nurses' gloves after the application or removal of a chemotherapy bag. Finally, the waterproof overshoes may have picked up contamination on the floor and potentially spread it. Overshoes are essential for protecting the surgeons' shoes but can spread contamination on the floor and hands when they are removed.

Biological measurements

Biological monitoring of occupational exposure is a preventive tool to assess and manage the chemical risks related to antineoplastic drugs. It is the best tool for internal contamination assessment since it allows to take into account at the level of each individual, all exposure pathways (respiratory, dermal, oral), the wearing or not of the individual protective equipment, their effectiveness, personal hygiene practices, gestures and professional practices, and antineoplastic drugs amount handled [START_REF] Aylward | Sources of variability in biomarker concentrations[END_REF][START_REF] Lepage | Occupational exposure to antineoplastic drugs: informations for biological monitoring[END_REF][START_REF] Lehman-Mckeenan | Absorption, distribution, and excretion of toxicants[END_REF].

In this study, no internal contamination was found in the ePIPAC staff even after implementing two successive ePIPAC procedures. These results showed the effectiveness of the individual and collective protective measures applied. Limits i) As reported by Ametsbichler et al. [START_REF] Ametsbichler | Occupational exposure to cisplatin/oxaliplatin during Pressurized Intraperitoneal Aerosol Chemotherapy (PIPAC)?[END_REF] we found some differences between individual surface contamination measurements after the two e-PIPAC procedures, which make interpretation of results difficult. Moreover, the threshold of detection depend to the analysis method used and can explain the differences of the results between the studies. Nevertheless, the health risk is influenced by the level and frequency of exposure, the toxicity of the drug, the existence of proper work practices.

ii) In our center, we perform all PIPAC and e-PIPAC procedures in the same OR. But, although the cleaning protocol is performed after each procedure, this can produce a bias because we have not analysed the Operate Room contamination before the first e-PIPAC A. Thus, as suggested by Ametsbichler et al. [START_REF] Ametsbichler | Occupational exposure to cisplatin/oxaliplatin during Pressurized Intraperitoneal Aerosol Chemotherapy (PIPAC)?[END_REF], there might have been a contamination before the beginning of this study.

iii) We cannot confirm definitively that the chemotherapy aerosols of e-PIPAC have contaminated the angioinjector and the floor. Indeed, the operator's gloves probably contaminated upon receipt of the perfusion bag may have been the cause of the screen contamination. Moreover, the overshoes have maybe spread this contamination and be the causes of the majority of the environmental contamination in our study. As this study was conducted on only two ePIPAC, further sampling studies are needed to confirm these results.

Conclusion

No traces of cisplatin were found in the air, and none contamination of cisplatin and doxorubicin was detected in healthcare workers and confirmed the effectiveness of the individual and collective protective measures applied. Although the ePIPAC safety and cleaning standards were implemented to reduce the environmental exposure to chemotherapy cisplatin and doxorubicin contamination was detected on the OR floor, surfaces, devices and personal protective equipment, which persisted after a standardized cleaning procedure.

Nevertheless, the contamination rate is estimated to be low, it is necessary to be vigilant during all stages of the ePIPAC procedure and to respect all checklists.

Figure 1 :Figure 2 :Figure 3 :Figure 4 :

 1234 Figure 1: Static samples from the filtered return-air grille (a) and the PIPAC injector,

Figure 5 :Annex 1 : 2 :

 512 Figure 5: Quantity of cisplatin (ng/wipe) in floor samples from the contaminated room

20 Legends Table 1 : Doxorubicin and cisplatin quantity measured in wipe samples collected after the ePIPAC procedure and after cleaning the operating room.Table 2 : Doxorubicin and cisplatin quantity measured in wipe samples from surfaces after the ePIPAC procedure.

 Legends12

Table 3 : Doxorubicin and cisplatin contamination of gloves, overshoes and a single-use microfiber mop after the ePIPAC procedures.Table 4 :

 34 Air contamination

1 =

 1 Quantity of cisplatin (ng/wipe) in floor samples from the contaminated room after ePIPAC A 2 = Quantity of cisplatin (ng/wipe) in floor samples from the clean room after ePIPAC A 3= Quantity of cisplatin (ng/wipe)in floor samples from the contaminated room after ePIPAC B 4 = Quantity of cisplatin (ng/wipe)in floor samples from the clean room after ePIPAC B

			Doxorubicin			Platinum
			pg/wipe (pg/cm 2 )			ng/wipe (ng/cm 2 )
		ePIPAC A	ePIPAC B	ePIPAC A		ePIPAC B
		After ePIPAC	After cleaning	After ePIPAC	After cleaning	After ePIPAC	After cleaning	After ePIPAC	After cleaning
	Under the head of the surgical table	ND	ND	6.7 (0.3)	6.3 (0.25)	20 (0.09)	20 (0.09)	50 (0.2)	700 (3.1)
	Under the operating table right side	ND	ND	5.7 (0.2)	ND	50 (0.2)	30 (0.13)	20 (0.09)	140 (0.6)
	Under the operating table left side	ND	7.2 (0.3)	6 (0.2)	ND	10 (0.04)	30 (0.13)	310 (1.38)	460 (2.0)
	Under the operating table legs	ND	ND	ND	ND	50 (0.2)	30 (0.13)	50 (0.2)	1 580 (9.1)
	Under the closed aerosol waste system	ND	Traces	ND	ND	30 (0.13)	40 (0.18)	140	1 760 (7.8)
	Floor under the PIPAC injector	ND	6.7 (0.3)	ND	ND	30 (0.13)	30 (0.13)	215 930	2 140 (9.5)

Table 1 :

 1 Doxorubicin and cisplatin quantity measured in wipe samples collected after the ePIPAC procedure and after cleaning the operating room. ND: not detected. Traces: concentration between the LOD and LOQ value of the analytical method.

	Doxorubicin pg/wipe	Platinum
	(pg/cm 2 )		ng/wipe (ng/cm 2 )
	ePIPAC A	ePIPAC B	ePIPAC A	ePIPAC B

Table 2 :

 2 Doxorubicin and cisplatin quantity measured in wipe samples from surfaces after the ePIPAC procedure. ND: not detected.

		Doxorubicin	Platinum
		pg/wipe	ng/device
		(pg/cm 2 )	(ng/cm 2 )
		ePIPAC A	ePIPAC B	ePIPAC A	ePIPAC B
	Surgeon's right overshoe	5.1 (0.2)	5.6 (0.2)	50 (0.3)	100 (14.3)
	Surgeon's left overshoe	ND	ND	100 (0.6)	550 (3.1)
	Nurse's right overshoe	ND	ND	150 (0.9)	11 800 (67.4)
	Nurse's left overshoe	ND	ND	200 (1.1)	6 100 (34.9)
	Second surgeon's right overshoe	ND	Traces	300 (1.7)	3 700 (21.1)
	Second surgeon's right overshoe	ND	ND	600 (3.4)	20 400 (116.6)
	Nurse's right glove fixing the perfusion bag (9/16)	650 (4.5)	ND	1 550 (10.8)	850 (5.9)
	Nurse's left glove fixing the perfusion bag	1 121 (7.9)	6.4 (0.04)	2 050 (14.2)	450 (3.1)

Surgeon's right glove closing the laparoscopic incisions after ePIPAC.

	54.8 (0.4)	20.8 (0.13)	1 600 (11.1)	550 (3.82)

Surgeon's left glove closing the laparoscopic incisions after ePIPAC

	Auxiliary nurse's left overshoe	6.1 (0.04)	ND	300 (2.1)	300 (2.1)
	Auxiliary nurse's right glove after cleaning the operating room	ND	ND	650 (4.5)	700 (4.8)
	Auxiliary nurse's left glove after cleaning the operating room	ND	ND	1 200 (8.3)	1 000 (6.9)
	Single-use microfiber mop	720.3	ND	3 050	16 950
		484.2 (3.4)	72 (0.5)	3 100 (21.5)	600 (4.2)
	Nurse's right glove cleaning the PIPAC injector	18.9 (0.13)	5.2 (0.04)	1 000 (6.9)	3 350 (23.3)
	Nurse's left glove cleaning the PIPAC injector	Traces	6.3 (0.04)	950 (6.6)	42 100 (292.4)
	Anaesthetist's right glove	13.3 (0.09)	6.7 (0.04)	1 400 (9.7)	1 650 (11.4)
	Anaesthetist's left glove	ND	5.1 (0.03)	1 850 (12.8)	2 750 (19.1)
	Auxiliary nurse's right overshoe	ND	5.6 (0.03)	250 (1.7)	600 (4.2)

Table 3 :

 3 Doxorubicin and cisplatin contamination of gloves, overshoes and a single-use microfiber mop after the ePIPAC procedures. ND: not detected.

		Platinum (ng/filter)	Air sampling time (min)	Airflow (L/min)	Air sampling volume (L)
		ePIPAC A	ePIPAC B ePIPAC A ePIPAC B ePIPAC A	ePIPAC B	ePIPAC A	ePIPAC B
	Surgeon's breathing zone	ND	ND	92	60	2.031	2.041	186.852	122.46
	Static sample on the
	PIPAC injector towards	ND	ND	40	25	2.041	2.027	81.64	50.675
	operating table
	Static sample near the filtered return-air grille	ND	ND	40	25	2.015	2.054	80.6	51.35

Table 4 :

 4 Air contamination. ND: not detected.

This project was also supported by funds from the Limoges Hospital committee (Public Hospital). This funder had no role in study design, data collection and analysis. authors declare no conflicts of interest. original draft. TAIBI Abdelkader: conceptualization, patient management, methodology, and writing and review of the original draft. CANAL-RAFFIN Mireille: conceptualization and methodology-review of the original draft. DURAND-None analysis was performed on patients, no ethics committee approval was required for this study.

data and material (data transparency): Not applicable Code availability (software application or custom code): Not applicable

Environmental measurements Air contamination

Inhalation of aerosols is dangerous situations for OR personnel. The air sampling method conducted in this study did not detect platinum during stationary or personal inhalable aerosol sampling. However, caution is still necessary because current sampling methods are not sufficiently optimized for standardized measurements. As there are no data on a suitable filter for detecting doxorubicin, the analysis was not performed for doxorubicin.

In most studies that investigated environmental exposure to chemotherapeutic drugs during PIPAC, air samples were evaluated [START_REF] Solass | Pressurized intraperitoneal aerosol chemotherapy (PIPAC): occupational health and safety aspects[END_REF][START_REF] Willaert | Occupational safety of pressurized intraperitoneal aerosol chemotherapy (PIPAC)[END_REF][START_REF] Oyais | Occupational Health Aspects of Pressurised Intraperitoneal Aerosol Chemotherapy (PIPAC): Confirmation of Harmlessness][END_REF][START_REF] Graversen | Environmental safety during the administration of Pressurized IntraPeritoneal Aerosol Chemotherapy (PIPAC)[END_REF]. Willaert et al. did not detect any trace of chemotherapeutic drugs in air samples collected from the breathing zones of the surgeon and anaesthesiologist after ePIPAC [START_REF] Willaert | Occupational safety of pressurized intraperitoneal aerosol chemotherapy (PIPAC)[END_REF]. Delhorme et al. reported no contamination of doxorubicin in ambient air samples after PIPAC [START_REF] Delhorme | Occupational safety of pressurized intraperitoneal aerosol chemotherapy (PIPAC) in an operating room without laminar airflow[END_REF]. As Delhorme et al. our study was conducted in an OR without unidirectional airflow [START_REF] Delhorme | Occupational safety of pressurized intraperitoneal aerosol chemotherapy (PIPAC) in an operating room without laminar airflow[END_REF]. It would seem that by respecting the set up previously described using single-use cover plastic sheets, absorbent mats and balloon trocars, security conditions are sufficient and the risk of exposure to the chemotherapeutic drugs was limited and the risk of exposure to the chemotherapeutic drugs by air rout was limited. Thus, cisplatin cannot be inhaled by OR personnel during ePIPAC if no accidental leakage occurs.

Surface contamination

In this study, the total amount of cisplatin contamination was much more important than that of doxorubicin contamination. Several reasons explain this difference of contamination level as the most important is that the used dose of cisplatin dose is usually five times higher than doxorubicin dose. Additionally, technical or human error regarding use of ePIPAC equipment