A Boussaroque

M.-P Audrézet

C Raynal

I Sermet-Gaudelus

T Bienvenu

C Férec

A Bergougnoux

M Lopez

V Scotet

A Munck

E Girodon

PENETRANCE IS A CRITICAL PARAMETER FOR ASSESSING THE DISEASE LIABILITY OF CFTR VARIANTS Running title: Variable penetrance associated with CFTR variants

Keywords: penetrance, CF, CFTR, newborn screening, inconclusive cases, CFSPID

Background: Major issues of newborn screening (NBS) for CF are the assessment of disease liability of variants and of the penetrance of clinical CF, notably in inconclusive diagnosis. The penetrance of CF is defined as the risk of a particular genotype to lead to a CF phenotype.

Methods:

We aimed to get insight into the penetrance of CF for fifteen CFTR variants: 5 frequent CF-causing and 10 classified as of varying clinical consequence (VCC) or associated with a CFTR-related disorder (CFTR-RD) in CFTR2 or CFTR-France databases. The penetrance was approached by: 1) comparison of variant allelic frequencies in CF patients (CFTR2) and in the general population; 2) estimation of the likelihood of a positive NBS test for the 14 compound heterozygous with F508del and the F508del homozygous genotypes, defined as the ratio of detected/expected number of neonates with a given genotype in the 2002-2017 period.

Results: A full penetrance was observed for severe CF-causing variants. Five variants were more frequently found in the general population than in CF patients: TG11T5, TG12T5, TG13T5, L997F and R117H;T7. The likelihood of a positive NBS test was 0.03% for TG11T5, 0.3% for TG12T5, 1.9% for TG13T5, 0.6% for L997F, 11.8% for D1152H, and 17.8% for R117H;T7.

Penetrance varied greatly for variants with discrepant classification between CFTR2 and CFTR-France: 5.1% for R117C, 12.3% for T338I, 43.5% for D110H and 52.6% for L206W.

Conclusion:

These results illustrate the contribution of genetics population data to assess the disease liability of variants for diagnosis and genetic counseling purposes.

Introduction

Newborn screening (NBS) for cystic fibrosis (CF) strives for the early detection of CF in infants to implement appropriate interventions and ultimately improve long term respiratory outcomes and survival [START_REF] Castellani | Cystic fibrosis diagnosis in newborns, children, and adults[END_REF][START_REF] Tridello | Early diagnosis from newborn screening maximises survival in severe cystic fibrosis[END_REF]. Beyond the goal of NBS, almost all protocols worldwide identify infants who screen positive with an inconclusive determination [START_REF] Barben | The expansion and performance of national newborn screening programmes for cystic fibrosis in Europe[END_REF], so called CFSPID (Cystic Fibrosis Screen Positive, Inconclusive Diagnosis) in EU or CRMS (CFTR-related metabolic syndrome) in the US. The harmonized definition published in 2017 includes infants with sweat chloride between 30-59 mmol/L and 0 or 1 CF-causing mutation, or a sweat chloride below 30 mmol/L and 2 CFTR mutations, at least one of which has unclear phenotypic consequences [START_REF] Farrell | CFTR-France, a national relational patient database for sharing genetic and phenotypic data associated with rare CFTR variants[END_REF]. The proportion of cases with an inconclusive diagnosis further increases in programmes that include extended CFTR gene sequencing as a part of the NBS strategy [START_REF] Barben | The expansion and performance of national newborn screening programmes for cystic fibrosis in Europe[END_REF]5]. It is thus a challenge to accurately assess the disease liability of variants, both for diagnosis and genetic counseling purposes. This requires to take into account available clinical information in databases and the literature, electrophysiological test results, functional analysis at the mRNA and protein level, and population genetics [6,7]. Databases may provide discordant information one should be aware of, notably because of different designs, so that the message given to the parents of a neonate may differ when referring to them. CFTR2 collects data from North American and European CF registries [8], i.e. only from patients diagnosed with CF. CFTR-France is dedicated to the interpretation of rare variants and is built on data from genetics laboratories and the French CF registry [9]. It collects genetic and clinical data from patients with CF and CFTR-related disorders (CFTR-RD) and from asymptomatic individuals who are compound heterozygous for two CFTR variants. Furthermore, the frequency of variants and genotypes in the general population should be considered to properly evaluate the risk of a particular genotype to cause clinical CF; that is, the penetrance of clinical CF in individuals carrying a given genotype. As an illustration, taking into account both clinical observations and epidemiological data, it was estimated that the penetrance of CF in individuals compound heterozygous for R117H;T7 and F508del was as low as 0.03% and that of CFTR-RD was 3% [START_REF] Thauvin-Robinet | The very low penetrance of cystic fibrosis for the R117H mutation: a reappraisal for genetic counselling and newborn screening[END_REF]. Using variant frequencies in the general population provided by whole exome or genome sequencing (such as ExAC or gnomAD [START_REF] Karczewski | Variation across 141,456 human exomes and genomes reveals the spectrum of loss-offunction intolerance across human protein-coding genes[END_REF]), we aimed to get insight into the penetrance of CF, associated with a set of CFTR variants which have been identified through NBS in France [START_REF] Audrézet | Comprehensive CFTR gene analysis of the French cystic fibrosis screened newborn cohort: implications for diagnosis, genetic counseling, and mutation-specific therapy[END_REF][START_REF] Munck | Optimization of the French cystic fibrosis newborn screening programme by a centralized tracking process[END_REF]. In this study, we showed that the low penetrance associated with some variants found in inconclusive cases might help the clinician to adapt medical care and follow-up of these newborns, as well as genetic counseling given to families.

Methods

In France, NBS for CF is a nationwide programme which relies on a 3-tiered strategy: i) IRT measurement at 3 days of age; ii) test for the most frequent CF-causing mutations in France (list in Table 1 caption, mutation detection rate at or above 80% in all regions) using the Elucigene® CF30v2 kit (Yourgene Health, Manchester, UK) when the IRT value is above the cut-off level; iii) retesting of IRT at 21 days of age if no mutation is identified in hypertrypsinogenemic infants with ultra-high IRT at day-3 (safetynet procedure) or if written consent for DNA testing is not obtained. Sweat testing is performed in CF care centers when one or two mutations are identified or in the absence of mutation when IRT at day-21 is above the cut-off level. Infants with a positive or intermediate sweat test are then subjected to an in-depth CFTR gene study, which mutation detection rate is 99.7% [START_REF] Audrézet | Comprehensive CFTR gene analysis of the French cystic fibrosis screened newborn cohort: implications for diagnosis, genetic counseling, and mutation-specific therapy[END_REF]. For the present study, we selected a set of 15 CFTR variants, comprising frequent CF-causing variants included in the French NBS panel as controls and variants frequently detected in inconclusive cases of NBS in France after extended CFTR gene analysis and which may have been differently classified by CFTR2 and CFTR-France databases (Table 1). They include (i) variants classified as "CFTR-RD-causing" in CFTR-France, which are observed in patients with a CFTR-RD when combined in trans with a CFcausing variant, and (ii) variants of varying clinical consequences (VCC), which are reported as well in CF patients as in patients with a CFTR-RD, when in trans with a CF-causing variant. In CFTR2, as the CFTR-RD class is not stated, VCC refers to variants observed in patients with CF and in patients who do not have CF.

Variants were described according to Human Genome Variation Society recommendations and to traditional names in Table 1. Traditional names were used in the text for better readability.

Their allelic frequency (p) in the general population was mostly taken from gnomAD in the European non-Finnish population [START_REF] Karczewski | Variation across 141,456 human exomes and genomes reveals the spectrum of loss-offunction intolerance across human protein-coding genes[END_REF]. GnomAD v2.1.1 collects exome and genome data from 125,748 and 15,708 unrelated individuals, respectively, who were enrolled in various diseasespecific and population genetic studies. Allelic frequencies of the most frequent variants were taken in the French population: in a sample of 10490 alleles for F508del and R117H [START_REF] Thauvin-Robinet | The very low penetrance of cystic fibrosis for the R117H mutation: a reappraisal for genetic counselling and newborn screening[END_REF] and in a sample of 4380 alleles for the TG11T5, TG12T5 and TG13T5 variants.

As an approach to assess the penetrance of CF, in a first step, allelic frequencies in the general population were compared with those observed in CF patients, taken from CFTR2. We then estimated the likelihood of a positive NBS test for the selected genotypes, all in combination with F508del. Based on variant allelic frequency in the general population (pVariant), the total number of neonates born in the 2002-2017 period (N) and assuming Hardy-Weinberg equilibrium, the number of infants born in France in the 2002-2017 period expected to carry a given variant in trans with F508del was equal to 2 x pF508del x pVariant x N. The expected number of F508del homozygous newborn infants expected was equal to pF508del 2 x N. The likelihood of a positive NBS test was then defined as the ratio of detected/expected number of neonates carrying a given genotype in the same period. Reliable sweat test data was only available for diagnosis for a subset of patients. For R117H, only data for the 2002-2014 period, when the mutation was still part of the NBS panel (Elucigene® CF30, note in Table 1), were taken into account for accurate comparison. Indeed, the number of neonates carrying R117H and detected at birth dramatically decreased since the removal of the variant in January 2015 [START_REF] Munck | Optimization of the French cystic fibrosis newborn screening programme by a centralized tracking process[END_REF].

Statistical analysis was carried out using the R statistical software (version 3.2.3). All tests were performed two-tailed at a level of significance of 5%. For each genotype, the likelihood of a positive NBS was estimated as described above and its 95% confidence interval (CI) was determined. The exact binomial test was used to compare the observed genotype frequency (i.e. the number of neonates carrying this genotype divided by the total number of neonates born in the period) with the expected frequency (estimated from database frequencies and assuming Hardy-Weinberg equilibrium).

Results

Of the 15 variants, five were classified as CF-causing and two as VCC in both databases (CFTR2 and CFTR-France). Discrepancies were observed for the other eight: CF in CFTR2 and VCC in CFTR-France (n=3); CF in CFTR2 and CFTR-RD in CFTR-France (n=1); VCC in CFTR2 and CFTR-RD in CFTR-France (n=3); and non-CF in CFTR2 and CFTR-RD in CFTR-France (n=1) (Table 1).

The five CF-causing variants (R334W, F508del, G542X, R553X and N1303K) were found 65 to 88 times more frequently in CF patients than in the general population. Five VCC or CFTR-RD variants according to CFTR-France (R117C, T338I, L206W, D1152H and D110H) were found 3 to 13 times more frequently in CF patients than in the general population. The last five variants had an allelic frequency in the general population higher than in CF patients: all three T5 variants (TG11T5, TG12T5 and TG13T5), L997F and R117H. In particular, TG11T5 was found up to 95 times more frequently in the general population than in CF patients.

The same trend was observed with estimates of likelihood of a positive NBS test which varied greatly (Table 1 and Figure 1). A total of 12,320,023 infants were born in France in the 2002-2017 period. Of these, 2,441 had two CFTR variants and were diagnosed with CF or reported inconclusive. 987 were homozygous for F508del and 355 carried F508del in trans with one of the other 14 variants included in this study. Of the 15 variants, G542X, R553X and N1303K were fully penetrant while the most common pathogenic variant, F508del, showed a reduced likelihood of a positive NBS test, evaluated at 71.3%. Meanwhile, all three T5 variants showed a likelihood of a positive NBS test below 2%.

There were less F508del homozygous nenonates observed (987) than expected (1384), providing a ratio of 71.3%. This significant difference should be regarded in the light of prenatal diagnoses and subsequent terminations of pregnancy (TOPs). Indeed, based on 2007-2017 data from the Agence de la biomédecine, who annually collects prenatal diagnosis activity from all molecular genetics laboratories in France, the number of TOPs for CF was 512 in this 11-year period [START_REF]Rapports d'activité médicale et scientifique de l'Agence de la biomédecine, diagnostic prénatal par génétique moléculaire[END_REF],

and extrapolated to 744 in the 2002-2017 total period. Based on a F508del allelic frequency of 67.24% in the French CF newborns [START_REF] Audrézet | Comprehensive CFTR gene analysis of the French cystic fibrosis screened newborn cohort: implications for diagnosis, genetic counseling, and mutation-specific therapy[END_REF] and thus assuming that 45.21% of the TOPs were made for F508del homozygous fetuses (that is, 336), a data which is however not available in the reports, this would bring the penetrance associated with the F508del homozygous genotype to 95.59%, which is not statistically different from 100%.

Discussion

Referral to clinical databases is most often the first step in the process for assessing the disease liability of variants identified in patients and asymptomatic individuals. Importantly, observations in CF patients only, i.e. CFTR2, represent the 'tip of the iceberg' of all possible phenotypes associated with a given genotype and CFTR-France may report a more substantial part of them. Other criteria should be taken into account, such as population, computational, functional and segregation data, according to international guidelines [START_REF] Richards | Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[END_REF][START_REF] Amendola | Performance of ACMG-AMP variant-interpretation guidelines among nine laboratories in the Clinical Sequencing Exploratory Research Consortium[END_REF]. We used population data to assess the penetrance associated with a panel of CFTR genotypes that have been identified in infants with a positive NBS result and an inconclusive diagnosis, and compared the results with those obtained for known CF-causing genotypes. It is important to note that the frequency of some variants found in gnomAD may not be used for accurate calculation, especially when differing from that of the local population.

As a reference, the allelic frequency of variants assessed as CF-causing in the two databases was 65 to 88 times higher in the population of CF patients than in the general population. As expected, the likelihood of a positive NBS test evaluated for CF-causing variants G542X, R553X and N1303K in trans of F508del was around 100%. The figure of 71.3% for the F508del homozygous genotype would be brought to 96% if considering TOPs following prenatal diagnoses, as documented above. The reduced likelihood of a positive NBS test of 58% associated with the CF-causing R334W variant could be explained by a residual function of the mutant protein [8,[START_REF] Sheppard | Mutations in CFTR associated with mild-disease-form Cl-channels with altered pore properties[END_REF], which is consistent with a mild form of CF as compared with other CFcausing genotypes.

Strikingly, the higher frequency of some variants in the general population as compared with the CF population, especially the T5 variants and L997F, is a strong argument against a severe pathogenic effect, suggesting a need for reclassification of these variants. The low penetrance of TG11T5 is consistent with that reported by Groman et al., where the variant was observed at higher frequency in fertile men than patients with congential bilateral absence of the vas deferens (CBAVD) [START_REF] Groman | Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign[END_REF]. The description of the T5 variants in CF patients and their classification in CFTR2 as VCC, may be partly explained by the existence of other pathogenic variants located on the same parental allele (i.e., in cis), in the context of complex alleles, such as numerous documented in CFTR-France [9]. Evidence for non-penetrance had also been documented for L997F, hence its classification as non CF-causing in CFTR2, because of its description in fathers of CF patients who carried in trans a previously accepted CF-causing variant [START_REF] Sosnay | Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene[END_REF].

An intermediate penetrance was observed for R117C, D110H and L206W, which have been reported as CF-causing in CFTR2 while VCC in CFTR-France, and for D1152H, reported as VCC in both databases. D110H, L206W and D1152H were found around 10 times more frequently in CF alleles (CFTR2) than in the general population and the likelihood of a positive NBS test in neonates carrying these variants in trans of F508del was around 50% for D110H and L206W, and 12.5% for D1152H. This disparity for D1152H is likely due to normal or intermediate sweat chloride values associated with this variant [START_REF] Burgel | Non-classic cystic fibrosis associated with D1152H CFTR mutation[END_REF]. The penetrance associated with R117C was even lower, being found only 3 times more frequently in CF patients than in the general population, with the likelihood of a positive NBS test being 5.5%. Although these data do not question the required follow-up of neonates detected with an inconclusive diagnosis, they could be considered to adapt infants care and frequency of medical visits, and eventually to shorten duration of follow-up [START_REF] Ren | Cystic Fibrosis Transmembrane Conductance Regulator-Related Metabolic Syndrome and Cystic Fibrosis Screen Positive, Inconclusive Diagnosis[END_REF][START_REF] Sermet-Gaudelus | Guidelines for the clinical management and follow-up of infants with inconclusive cystic fibrosis diagnosis through newborn screening[END_REF]. These data should also be used to modulate the information given to the parents, in terms of variable phenotype and of genetic counselling. They could also be considered in decision making regarding mutation-guided therapy, which is avaible notably for R117C, L206W and D1152H. Moreover, although not assessed in this study, the actual risk of developing symptoms compatible with CF or CFTR-RD is probably lower, as shown for R117H;T7. Indeed, considering the 2002-2014 period before withdrawal of R117H from the NBS panel, the likelihood of a positive NBS test was assessed at 17.81%. Compared with the actual penetrance of 0.03% for CF and of 3% for CFTR-RD in individuals compound heterozygous for R117H;T7 and F508del [START_REF] Thauvin-Robinet | The very low penetrance of cystic fibrosis for the R117H mutation: a reappraisal for genetic counselling and newborn screening[END_REF], these data further show that this genotype is selected by NBS for CF, with the majority of cases expected to be asymptomatic throughout their lifetime or not symptomatic before adulthood.

In conclusion, taking into account the frequencies of variants in the general population is critical to assess the disease liability of variants, especially in asymptomatic conditions and this emphasizes the utility of population genetic databases such as gnomAD. Our study shows that a number of variants identified in inconclusive cases of NBS for CF in asymptomatic infants are associated with a low or very low likelihood of a positive NBS test. These data stress the importance to document the penetrance of variants included in NBS panels and argue against the implementation of CFTR gene extended sequencing as a second-tier test after immuno-reactive trypsinogen measurement. Indeed, this would lead to a huge increase of inconclusive cases who would not develop CF during their lifetime, carrying variants of VCC or of unknown significance. These data are also of utmost importance in the era of increasing requests for genetic testing in the general population, either in a preconceptional setting, i.e. CF carrier testing in partners of CF carriers and in couples without any family history of CF, or in the Foundation. J Pediatr. 2017 Feb;181S:S4-S15.e1. doi: 10.1016/j.jpeds.2016.09.064. Erratum in: J Pediatr. 2017 May;184:243. PubMed PMID: 28129811.

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 5 Kharrazi M, Yang J, Bishop T, Lessing S, Young S, Graham S, Pearl M, Chow H, Ho T, Currier R, Gaffney L, Feuchtbaum L; California Cystic Fibrosis Newborn Screening Consortium. Newborn screening for cystic fibrosis in California. Pediatrics. 2015 Dec;136(6):1062-72. doi: 10.1542/peds.2015-0811. Epub 2015 Nov 16. PubMed PMID: 26574590. [6] Castellani C, Cuppens H, Macek M Jr, Cassiman JJ, Kerem E, Durie P, Tullis E, Assael BM, Bombieri C, Brown A, Casals T, Claustres M, Cutting GR, Dequeker E, Dodge J, Doull I, Farrell P, Ferec C, Girodon E, Johannesson M, Kerem B, Knowles M, Munck A, Pignatti PF, Radojkovic D, Rizzotti P, Schwarz M, Stuhrmann M, Tzetis M, Zielenski J, Elborn JS. Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice. J Cyst Fibros. 2008 May;7(3):179-96. doi: 10.1016/j.jcf.2008.03.009. Review. PubMed PMID: 18456578; PubMed Central PMCID: PMC2810954. [7] Sosnay PR, Salinas DB, White TB, Ren CL, Farrell PM, Raraigh KS, Girodon E, Castellani C. Applying cystic fibrosis transmembrane conductance regulator genetics and CFTR2 /j.jpeds.2016.09.063. PubMed PMID: 28129809. [8] US CF Foundation, Johns Hopkins University, The Hospital for Sick Children, Baltimore. The Clinical and Functional TRanslation of CFTR (CFTR2). Available from: http://cftr2.org.; 2019 [updated 10/01/2020; cited 2011]. [9] Claustres M, Thèze C, des Georges M, Baux D, Girodon E, Bienvenu T, Audrezet MP, Dugueperoux I, Férec C, Lalau G, Pagin A, Kitzis A, Thoreau V, Gaston V, Bieth E,

Table 1 :

 1 Epidemiological and penetrance data associated to CFTR variants : not documented; NN: neonates; * refers to allelic frequencies in the French general population; # c.350G>A (R117H) is no longer part of the French NBS panel as of January 2015. The data were thus collected during the 2002-2014 period with 10 046 679 NN. ‡ Value set at 100% because observed was greater than expected; Variants included in the French NBS panel are indicated in bold. W846X. NBS panel Elucigene®CF30v2 as of January 2015: same panel as above without R117H.Figure 1: Likelihood of a positive NBS test on a scale for genotypes combining F508del and one of the 15 variants * Statistically significant difference between detected and expected number of neonates carrying a given genotype.# For the F508del homozygous genotype, correction with the expected number of termination of pregnancies after prenatal diagnoses was taken into account.

	Variant name		Phenotypic classification	Allele frequency
	cDNA (HGVS)	Protein (HGVS)	Legacy	CFTR2	CFTR-France	General population (gnomAD/ French*)	CF patients CFTR2 (n=142,036 )	Ratio General population/ CFTR2 frequency	Ratio CFTR2/ general population frequency	No. NN expected F508del/variant (n=12320023 NN)	No. NN detected (2002-2017 period)	Ratio detected/ expected	95% IC (%)	Likelihood of a positive NBS test	Significance (Exact binomial test)
	c.[1210-34_1210-6TG[11]T[5] p.(=)	TG11T5	VCC	CFTR-RD	0.0234*	0.00025	94.96	0.01	6111.7	2	0.0003	0.0003 to 0.0003	0.03%	<0.0001
	c.[1210-34_1210-6TG[12]T[5] p.(=)	TG12T5	VCC	CFTR-RD	0.0098*	0.00128	7.65	0.13	2559.6	9	0.0035	0.0035 to 0.0035	0.35%	<0.0001
	c.[1210-34_1210-6TG[13]T[5] p.(=)	TG13T5	VCC	VCC	0.0008*	0.00030	2.64	0.38	208.9	4	0.0191	0.0191 to 0.0191	1.91%	<0.0001
	c.2991G>C	p.Leu997Phe	L997F	Non CF	CFTR-RD	0.002544	0.00077	3.28	0.30	664.5	4	0.0060	0.0060 to 0.0061	0.60%	<0.0001
	c.349C>T	p.Arg117Cys	R117C	CF	VCC	0.0003724	0.00103	0.36	2.76	97.3	5	0.0514	0.0513 to 0.0515	5.14%	<0.0001
	c.3454G>C	p.Asp1152His D1152H	VCC	VCC	0.0003261	0.00402	0.08	12.33	85.2	10	0.1174	0.1172 to 0.1176	11.74%	<0.0001
	c.1013C>T	p.Thr338Ile	T338I	CF	CFTR-RD	0.00006201	0.00037	0.17	5.97	16.2	2	0.1235	0.1233 to 0.1237	12.35%	<0.0001
	c.[350G>A;1210-12[7]]	p.Arg117His	R117H;T7#	VCC	CFTR-RD	0.0029*	0.00088	3.30	0.30	617.7	110#	0.1780	0.1779 to 0.1783	17.81%	<0.0001
	c.328G>C	p.Asp110His	D110H	CF	VCC	0.00003522	0.00046	0.08	13.00	9.2	4	0.4348	0.4346 to 0.4351	43.48%	0,0969
	c.617T>G	p.Leu206Trp	L206W	CF	VCC	0.00024	0.00234	0.10	9.77	62.7	33	0.52645	0.5262 to 0.5267	52.64%	<0.0001
	c.1000C>T	p.Arg334Trp R334W	CF	CF	0.00004652	0.00302	0.02	64.93	12.2	7	0.5761	0.5758 to 0.5764	57.61%	0.1928
	c.1521_1523del	p.Phe508del	F508del	CF	CF	0.0106*	0.69744	0.02	65.78	1384.3	987	0.7130	0.7128 to 0.7133	71.30%	<0.0001
	c.1624G>T	p.Gly542Ter	G542X	CF	CF	0.0003571	0.02542	0.01	71.17	93.3	85	0.9113	0.9112 to 0.9115	91.13%	0.437
	c.3909C>G	p.Asn1303Lys N1303K	CF	CF	0.0001796	0.01581	0.01	88.03	46.9	47	1.0019	1.0019 to 1.0020	100% ‡	0.9418
	c.1657C>T	p.Arg553Ter	R553X	CF	CF	0.0001243	0.00931	0.01	74.90	32.5	33	1.0164	1.0164 to 1.0165	100% ‡	0.8608
	Note: NBS panel Elucigene®CF30 (legacy nomenclature): F508del, I507del, 1078delT, 1717-1G>A, 2183AA>G, 3659delC, 3849+10kbC>T, 621+1G>T, A455E,
	E60X, G542X, G551D, N1303K, R1162X, R117H, R334W, R347P, R553X, S1251N, W1282X, 1811+1,6kbA>G, 2789+5G>A, 3120+1G>A, 3272-26A>G,
	394delTT, 711+1G>T, G85E, Y1092X, Y122X,

ND

perspective of extended NBS for numerous genetic diseases by using wide genome analysis [START_REF] Ceyhan-Birsoy | Interpretation of Genomic Sequencing Results in Healthy and Ill Newborns: Results from the BabySeq Project[END_REF][START_REF] Kingsmore | Newborn testing and screening by whole-genome sequencing[END_REF].

Conflict of interest statement: none


Cai Zhang

Baiping Liu

Jodi Pawluski

Harry W M Steinbusch

Kirthana Kunikullaya

Cai Song
email: cai.song@dal.ca

The effect of chronic stress on behaviors, inflammation and lymphocyte subtypes in male and female rats

Keywords: depression, chronic stress, inflammation, lymphocytes, sex differences

Excessively released proinflammatory mediators from activated macrophages and lymphocytes may contribute to the etiology of depression. However, the relationship between lymphocytes and depression is not fully understood. Although women have higher depression risk than men, sex/gender differences in psychoneuroimmunological mechanisms are still unclear. To explore these two questions, chronic unpredictable mild stress (CUMS) was used to evaluate the changes in behaviors, inflammation and lymphocyte subtypes in adult male and female Wistar rats.

Results show that CUMS increased anhedonia and anxiety-like behaviors, along J o u r n a l P r e -p r o o f with increased serum corticosterone, hippocampal pro-inflammatory factors, CD11b, IFN-γ, IL-6 and IL-17, but decreased CD4, CD25, CD4/CD8 ratio, GFAP, 5-hydroxytryptamine (5-HT) and NE concentrations, regardless of sex. There was no positive correlation between sucrose preference and blood CD4/CD8 ratio, but a positive correlation between sucrose preference and spleen CD25, sucrose preference and neurotransmitters (NE and 5-HT), spleen CD25 and serum TGF-β1/IL-6 ratio were found, regardless of sex. Females presented higher basal locomotion, blood CD4, CD4/CD8 ratio, serum corticosteroid and IL-6 concentrations, but lower hippocampal norepinephrine (NE) than males. Although CUMS didn't induce significant sex differences, females presented more changes in CD4 and CD8 lymphocytes than male rats. CUMS caused abnormalities in corticosteroid, lymphocytes, cytokines and neurotransmitters, which might be the precursors for inducing depression-like behaviors in both sexes.

Introduction

Epidemiological studies have reported that women are more vulnerable to stress and it's effects on mental health compared to men [1], and are twice as likely as men to suffer from depression [START_REF] Marcus | Gender differences in depression: findings from the STAR*D study[END_REF]. Given the apparent sex difference in depression, it is unfortunate that most pre-clinical animal experiments have been carried out on male rodents [START_REF] Klein | Opinion: Sex inclusion in basic research drives discovery[END_REF]. Thus, it is important to explore sex/gender differences in depression, which may provide better understanding and treatment for this disease in males and females.

Chronic stress is often considered as an inducer of depression [START_REF] Heim | The role of early adverse life events in the etiology of depression and posttraumatic stress disorder. Focus on corticotropin-releasing factor[END_REF][START_REF] Kessler | The effects of stressful life events on depression[END_REF]. Consistent with the hypothalamic-pituitary-adrenal (HPA) axis and macrophage/T-lymphocyte hypothesis of depression, chronic stress damages the feedback of HPA axis and induces an abnormal increase of corticosteroid, which desensitizes glucocorticoid J o u r n a l P r e -p r o o f receptors located on lymphocytes and causes lymphocyte dysfunction [START_REF] Maes | The monocyte-T-lymphocyte hypothesis of major depression[END_REF][START_REF] Leonard | The immune system, depression and the action of antidepressants[END_REF]. In the past three decades, growing evidence indicates that activated macrophages and T lymphocytes-induced inflammatory responses may trigger the onset and development of depression [START_REF] Maes | The monocyte-T-lymphocyte hypothesis of major depression[END_REF][START_REF] Miller | Depression and immunity: a role for T cells?[END_REF] ( Fig 1). Meta-analyses indicate clinical depression is associated with significant alterations in toxic CD8 cells, helper CD4 and related subtypes, such as pro-inflammatory Th1 and Th17, as well as anti-inflammatory Th2 and Treg [START_REF] Shi | T cell responses in depressed mice induced by chronic unpredictable mild stress[END_REF][START_REF] Szałach | The Influence of Antidepressants on the Immune System[END_REF][START_REF] Herbert | Depression and immunity: a meta-analytic review[END_REF].

Chronic stress activates peripheral lymphocyte subtypes and central microglia to continuously secrete pro-inflammatory Th1/M1 cytokines interferon-γ (IFN-γ) and reduce anti-inflammatory Th2/M2 cytokines IL-4 [START_REF] Zhang | Minocycline ameliorates depressive behaviors and neuro-immune dysfunction induced by chronic unpredictable mild stress in the rat[END_REF][START_REF] Reiche | Stress and depression-induced immune dysfunction: implications for the development and progression of cancer[END_REF]. Furthermore, CD25 Treg lymphocytes derived from CD4 cells can suppress the development of pro-inflammatory factors and are beneficial to the treatment of major depression [START_REF] Obermanns | Analysis of cytokine levels, T regulatory cells and serotonin content in patients with depression[END_REF].

More importantly, in clinical trials, decreased pro-inflammatory IL-1β and IL-6 cytokines, and increased CD25 Tregs and CD4/CD8 ratio are markers of major depression recovery [START_REF] Himmerich | Regulatory T cells increased while IL-1β decreased during antidepressant therapy[END_REF][START_REF] Kubera | Leukocyte subsets in treatment-resistant major depression[END_REF]. However, some researchers report that chronic stress fails to induce the changes in blood CD4 and CD8 in female mice [START_REF] Silberman | Effects of chronic mild stress on lymphocyte proliferative response. Participation of serum thyroid hormones and corticosterone[END_REF], whereas a reduction of spleen CD4 lymphocyte, and some increases in serum IL-1β and IL-6, as well as hippocampal IL-6 and IL-4, are found in male mice [START_REF] Shi | T cell responses in depressed mice induced by chronic unpredictable mild stress[END_REF]. The fact that women are more likely to suffer from depression than men may be related to their greater sensitivity to stress and inflammatory stimuli [START_REF] Lin | Sex differences in the effects of acute and chronic stress and recovery after long-term stress on stress-related brain regions of rats, Cerebral cortex[END_REF][START_REF] Slavich | Stress, sex hormones, inflammation, and major depressive disorder: Extending Social Signal Transduction Theory of Depression to account for sex differences in mood disorders[END_REF]. However, chronic stress-induced changes in lymphocyte subtypes, as well as the relationship between lymphocytes and inflammatory factors in different sexes are still unclear.

In animal models, chronic unpredictable mild stress (CUMS) is used as a valid model of depression [START_REF] Heim | The role of early adverse life events in the etiology of depression and posttraumatic stress disorder. Focus on corticotropin-releasing factor[END_REF][START_REF] Kessler | The effects of stressful life events on depression[END_REF]. CUMS can directly activate M1-type microglia to produce pro-inflammatory mediators [START_REF] Carrillo-De Sauvage | Potent and multiple regulatory actions of microglial glucocorticoid receptors during CNS inflammation[END_REF], which impairs astrocyte and neuronal function, thereby reducing neurotrophin levels, monoamine neurotransmitter concentration and neurogenesis in the hippocampus, a key brain region implicated in mood and cognition [START_REF] Kreisel | Dynamic microglial alterations underlie stress-induced depressive-like behavior and suppressed neurogenesis[END_REF][START_REF] Macqueen | The hippocampus in major depression: evidence for the convergence of the bench and bedside in psychiatric research?[END_REF]. Furthermore, astrocytes can present neuroprotective and neuroinflammatory functions which are dependent on the microenvironment and polarizations of M1-and M2-type microglia [START_REF] Jha | Functional polarization of neuroglia: Implications in neuroinflammation and neurological disorders[END_REF]. For example, pro-inflammatory factors down-regulate the astrocyte marker glial fibrillary acidic protein (GFAP) and J o u r n a l P r e -p r o o f neurotrophin (BDNF) expression, and this can be reversed with TNF-α inhibitor infliximab treatment [START_REF] Şahin | TNF-alpha inhibition prevents cognitive decline and maintains hippocampal BDNF levels in the unpredictable chronic mild stress rat model of depression[END_REF]. Our previous studies, in addition to those of others, have also demonstrated that the M1-type microglia inhibitor, minocycline, can reverse CUMS-induced depression-like behaviors by balancing the pro-and anti-inflammatory response of microglia, and subsequently restoring astrocyte neurotrophic and neurotransmitter function [START_REF] Zhang | Minocycline ameliorates depressive behaviors and neuro-immune dysfunction induced by chronic unpredictable mild stress in the rat[END_REF][START_REF] Bassett | Minocycline alleviates depression-like symptoms by rescuing decrease in neurogenesis in dorsal hippocampus via blocking microglia activation/phagocytosis[END_REF]. Moreover, when compared to males, females were more vulnerable to inflammation-induced mood and behavioral changes [START_REF] Derry | Sex Differences in Depression: Does Inflammation Play a Role?[END_REF], with more decreased hippocampal serotonergic activity after CUMS [START_REF] Dalla | Chronic mild stress impact: are females more vulnerable?[END_REF].

Even though CUMS induces some changes in lymphocyte subtypes, glia phenotypes, inflammation and neurotransmitter function, the sex/gender differences in these changes, the relationship among behaviors, lymphocyte subtypes, inflammation and neurotransmitters in depression still remain unclear. Moreover, the hippocampus, controlling cognition and emotion, is a highly stress-sensitive brain region, which regulates behavioral and neuroendocrine responses in depression [START_REF] Lisman | Viewpoints: how the hippocampus contributes to memory, navigation and cognition[END_REF][START_REF] Sheline | The hippocampus and depression[END_REF]. Therefore, the current study used a CUMS model of depression for 49 days to determine if there are differences between male and female rats in depression-like behaviors, blood and splenic lymphocytes, serum and hippocampal inflammatory levels, and hippocampal glia cells and neurotransmitter's function. It was expected the sex would play a significant role in CUMS effects on behaviors, inflammatory measures and lymphocytes.

Materials and Methods

Animals

Forty adult male and female Wistar rats (8 weeks of age, Changsha Tianqin Biotechnology Co., Ltd) were housed two per cage with a 12 h light/dark cycle (lights on at 7:00 and off at 19:00) at the Guangdong Ocean University animal facility (SYXK (Yue) 2014-0053). The room temperature was set at 23 ± 1℃, humidity 50 ± 10%. Animals had standard rodent chow and water available ad libitum. Prior to the J o u r n a l P r e -p r o o f test procedure, rats were acclimated to the laboratory for seven days. The experiments were conducted in a blinded fashion and were approved by the Institutional Bioethics Committee (Guangdong Ocean University, China, IACUC-20170325-012). The experimental procedures were conducted in full compliance with the National Institutes of Health Guide for the Care and Use of the Laboratory Animals.

Experimental procedure

Twenty male and twenty female rats were divided equally into 4 experimental groups (n=10 in each group) as a) male control (Male + CT), b) male stress (Male + CUMS), c) female control (Female + CT), d) female stress (Female + CUMS).

2.3.Stress exposure paradigms

The CUMS protocol was carried out as previously described [START_REF] Zhang | Minocycline ameliorates depressive behaviors and neuro-immune dysfunction induced by chronic unpredictable mild stress in the rat[END_REF]. Briefly, 14 different stressors were applied (2 stressors per day) in a random order over a period of 49 days (Table 1).

Sucrose preference test

Anhedonia behavior was measured by the sucrose preference test as previously described [START_REF] Zhang | Minocycline ameliorates depressive behaviors and neuro-immune dysfunction induced by chronic unpredictable mild stress in the rat[END_REF]. On day 1, rats were offered two bottles of 1% (w/v) sucrose to consume at 19:00 for 24h. On day 2, rats were offered one bottle of 1% sucrose and one bottle of fresh water at 19:00 for 24h. On day 3, food and water deprivation occurred at 19:00 for 24h. On day 4, rats were offered one bottle of 1% sucrose and one bottle of fresh water at 19:00, and then the consumption of sucrose and fresh water after 1 h was weighed. Sucrose preference (SP) was calculated according to the following formula, SP = sucrose intake / (sucrose intake + water intake) × 100 %.

Elevated plus maze test

Anxiety-related behavior was measured with the elevated plus maze test as described previously [START_REF] Zhang | Minocycline ameliorates depressive behaviors and neuro-immune dysfunction induced by chronic unpredictable mild stress in the rat[END_REF]. Briefly, rats were placed on the central platform facing the same open arm and anxiety-related behaviors were recorded by SuperMaze behavior analysis system (Shanghai Xinruan Information Technology Co., Ltd, Shanghai, China) for 5 minutes by two highly trained observers blinded to the groups. Before

Open field test

Exploration and anxiety-related behaviors were assessed with the open-field test as described previously [START_REF] Zhang | Minocycline ameliorates anxiety-related self-grooming behaviors and alters hippocampal neuroinflammation, GABA and serum cholesterol levels in female Sprague-Dawley rats subjected to chronic unpredictable mild stress[END_REF]. Briefly, a 60W white light bulb was positioned 100 cm above the center of the cylindrical apparatus with white inner wall (diameter 1 m, height 40 cm). A rat was placed with it's head facing towards the same wall of the apparatus and the behavioral activity was recorded using the SuperMaze behavior analysis system (Shanghai Xinruan Information Technology Co., Ltd, Shanghai, China) for 5 min by two highly trained observers blinded to the groups. Before the test, rats were acclimatized to the room for 1 h. After the test the arena was cleaned with 0.5% EtOH. The number of entries into the center and the time spent in the central zone, as well as total locomotion in the open-field box, were recorded.

Collection of samples and assays

One day after behavioral testing, the animals were rapidly decapitated.

Immediately after decapitation, blood samples from the trunk (200 uL per rat) were

Quantitation of T-lymphocyte subtypes with flow cytometry

The blood and spleen were harvested after the rats were euthanized.

Lymphocytes were immediately extracted from the spleen by Lymphocyte Separation (Zhejiang Bozhen Biotechnology Co., Ltd, China) was added into blood samples to lyse erythrocytes. After washing with phosphate-buffered saline (PBS) buffer, the samples were detected and analyzed by CytoFLEX (Beckman, China).

Western blots

According to manufacturer's instructions, the total protein of the hippocampus was extracted with a commercial kit (BCA kit, Beijing dingguo changsheng Co., Ltd, China). Subsequently, 30-50 μg protein was loaded onto a 10% polyacrylamide gel, and then transferred to PVDF (Millipore, China) membrane. After incubation with western blot blocking solution reagent (Millipore, China) at room temperature for 2 hour, membranes were incubated with primary antibody overnight at 4 ℃. After washing, the membranes were further incubated with secondary antibody for 2 hours, then washed and detection was done using enhanced chemiluminescence (Millipore, China). The bands were scanned and analyzed using a chemiluminescence system (Tanon 5200, Shanghai, China). The antibodies for CD11b (ab75476, 160 kDa, 1:800) and BDNF (ab108319, 15 kDa, 1:2000) were purchased from Abcam Trading Company Ltd (Shanghai, China) and origin antibodies for GFAP (sc33673, 50 kDa, 1:400) and β-Actin (sc47778, 43 kDa, 1:400) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All target proteins were quantified by normalizing them to β-actin re-probed on the same membrane and then calculated as a percentage of the male control group.

HPLC analysis of neurotransmitters and metabolites

Hippocampal tissues were homogenized in 0.60 M ice-cold perchloric acid containing 50 mM Na 2 EDTA with 100 ng isoproterenol as an internal standard. The homogenates were centrifuged twice at 14,000 rpm/min for 15 min at 4℃.

Supernatants were filtrated with 0.45 μm membrane and transferred to new tubes. The

Results

CUMS induced anhedonia and decreased body weight, regardless of sex

There was a significant main effect of CUMS on sucrose consumption (F 1,36 =24.219, P<0.01) with CUMS significantly reducing sucrose consumption in both male and female rats (Fig. 2A), regardless of sex. Repeated ANOVA showed a significant interaction between group and time (weeks) on body weight (F 7, 36 = 15.775, P<0.01). The post-hoc test revealed that CUMS significantly decreased the body weight after five weeks in both sexes (Fig. 2B).

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CUMS and sex effects on locomotion in the open field test

There were significant main effects of CUMS (F 1,36 =6.782, P<0.05) and sex (F 1,36 =43.26, P<0.01) on locomotion, but no interaction effect (Fig. 3A). CUMS significantly decreased locomotion in both sexes with females having increased locomotion compared to males. After CUMS, the locomotion was reduced by 13.1%

in males and by 8.7% in females (Fig. 3A). Further, there was a significant main effect of CUMS on the number of central entries (F 1,36 =16.235, P<0.01) and the time in the center of the test (F 1,36 =18.363, P<0.01), regardless of sex and in the absence of an interaction effect (Fig. 3B,C). CUMS significantly decreased the number of entries and time spent in the central zone in both sexes.

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CUMS significantly induced anxiety-like behavior, regardless of sex

There

CUMS and sex significantly affect serum corticosterone level

Kruskal-Wallis test indicated that there was a significant changes in serum corticosterone (CORT) concentration (H=17.463, P<0.01). The post hoc test revealed serum corticosterone concentration at sacrifice was significantly higher in females than males in the control situation (P<0.05). Stressed animals had a significant increase in serum concentration of CORT at the time of sacrifice, regardless of sex (P<0.05). Compared to control animals of the same sex, CORT concentration at the time of sacrifice was increased by 14.5% in male and 18.3% in female rats exposed to CUMS (Fig. 5A). Moreover, the sex difference in locomotion behavior was reported by a previous study, in which increased locomotion was suppressed by corticosterone synthesis inhibitor metyrapone [START_REF] Ebada | Corticosterone and dopamine D2/D3 receptors mediate the motivation for voluntary wheel running in C57BL/6J mice[END_REF]. Hence, the correlation between corticosterone and locomotion was analyzed, and found a significant positive correlation between J o u r n a l P r e -p r o o f locomotion and serum CORT levels at sacrifice (r=0.4704, P<0.01), regardless of sex (Fig. 5B).

CUMS and sex effects on peripheral T-lymphocyte subtypes

There was a significant interaction effect between CUMS and sex in the percentage of lymphocyte CD4 (F 1,20 =5.983, P<0.05), CD8 (F 1,20 =4.473, P<0.05) and the ratio of CD4/CD8 (F 1,20 =6.143, P<0.05) in the blood (Fig. 6A,B,D). The post hoc tests revealed that lower blood CD8 percent (P<0.05) but higher CD4 percent (P<0.05) and CD4/CD8 ratio (P<0.05) were evident in female but not males in the control condition. Furthermore, stressed female rats showed a larger difference than stressed males when compared to control animals of the same sex. CD4 and CD4/CD8 ratio was decreased by 0.6% and 5.2% in male and 9.4% and 34.4% in female rats, but CD8 was increased by 2.8% in male and 36.2% in female rats after exposed to CUMS (Fig. 6B,D). There was also a significant main effect of CUMS, with stress significantly decreased spleen CD25 lymphocyte percentage in both sexes (F 1,20 =14.151, P<0.01) (Fig. 6C). Moreover, there was a significant positive correlation between spleen CD25 percentage and sucrose preference (r=0.6723, J o u r n a l P r e -p r o o f P<0.01) (Fig. 6F), but no correlations between blood CD4/8 ratio and sucrose preference (r=0.342, P=0.1018) (Fig. 6E), regardless of sex. Females vs. males.

CUMS and sex effects on serum pro-and anti-inflammatory factors

CUMS significantly elevated the concentration of IL-6 (F 1,28 =5.575, P<0.05) and IL-17 (F 1,28 =38.572, P<0.01) and decreased IL-10 (F 1,28 =24.756, P<0.01) and TGF-β1/IL-6 ratio (F 1,28 =22.577, P<0.01) in serum, regardless of sex (Fig. 7A,B,D,E). There was a significant effect of sex on serum levels of IL-6 (F 1,28 =16.32, P<0.01)

with females showing higher serum IL-6 concentrations than males (Fig. 7B). After CUMS exposure, male IL-6 levels were increased 22.5% than control males, and female IL-6 levels were 20.3% higher than control females (Fig. 7B). Kruskal-Wallis tests indicated that there were significant effects in serum IFN-γ (H=12.815, P<0.01)

and IFN-γ/IL-10 ratio (H=19.647, P<0.01). The post hoc test revealed that CUMS J o u r n a l P r e -p r o o f significantly increased serum IFN-γ (P<0.01) and IFN-γ/IL-10 ratio (P<0.01) in both sexes (Fig. 7A,C). Furthermore, this study found a positive correlation between CD25 and sucrose preference. A previous study has demonstrated that CD25 Treg lymphocytes was promoted by TGF-β, but inhibited by IL-6 [START_REF] Grosse | Circulating cytotoxic T cells and natural killer cells as potential predictors for antidepressant response in melancholic depression. Restoration of T regulatory cell populations after antidepressant therapy[END_REF]. In this case, there was a significant positive correlation between spleen CD25 and the ratio of TGF-β1/IL-6 in serum (r=0.4596, P<0.05) regardless of sex (Fig. 7F), which indicates a closed relationship between lymphocytes and inflammation in depression. J o u r n a l P r e -p r o o f

CUMS changed markers of microglia (CD11b) and astrocyte (GFAP) expression in the hippocampus, regardless of sex

CUMS significantly decreased the expression of hippocampal GFAP (F 1,20 =41.751, P<0.01) in both sexes (Fig. 8B) and there was no main effect of sex or an interaction effect. Kruskal-Wallis test indicated that there was a significant changes in hippocampal microglia CD11b expression (H=12.847, P<0.01). The post hoc test revealed CUMS significantly increased microglial protein expression of CD11b in both sexes (Fig. 8A).

CUMS and sex effects on hippocampal neurotransmitter

There was a significant main effect of CUMS (F 1,20 =30.466, P<0.01) and sex (F 1,20 =28.884, P<0.01) on hippocampal NE concentration without interaction effect (Fig. 10A). CUMS resulted in a reduction of hippocampal NE concentration by 21.6% in males and 20.2% in females compared to controls of the same sex (Fig. 10A).

Additionally, CUMS significantly reduced hippocampal 5-HT (F 1,20 =20.586, P<0.01) concentration (Fig. 10B), regardless of sex and there was no interaction effect. Moreover, positive correlations between sucrose preference and hippocampal NE (r=0.4195, P<0.05) (Fig. 10C), and hippocampal 5-HT (r=0.5943, P<0.01) was found (Fig. 10D).

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Discussion

In this study, sex differences and underlying neuroimmunological mechanisms in CUMS-induced depression using rat models was investigated. CUMS significantly induced depressive behaviors, increased serum corticosteroids and systemic pro-inflammatory response, decreased hippocampal neurotransmitter levels and peripheral lymphocyte function in both sexes. In unstressed animals sex differences were evident with females having increased levels of locomotion, serum CD4/CD8 ratio, serum CORT levels and serum IL-6 levels, as well as decreased hippocampal NE concentration compared to males. Although there were no significant sex differences, females presented more changes in CD4 and CD8 lymphocytes than males after chronic stress.

CUMS-induced changes in behaviors and serum corticosterone concentration

According to the HPA axis hypothesis of depression, excess release of glucocorticoids is one of the chief causes for mood disorders [START_REF] Checkley | The neuroendocrinology of depression and chronic stress[END_REF], and normalizing this by using a corticosteroid receptor antagonist could improve depression-like behaviors in stressed rats [START_REF] Liu | Effects of ginsenosides on hypothalamic-pituitary-adrenal function and brain-derived neurotrophic factor in rats exposed to chronic unpredictable mild stress[END_REF]. With regards to the sex differences, the present study found higher basal corticosteroid concentration and higher locomotor behaviors

(evidenced by open field test) in females compared to males. A previous study has reported that running exercise significantly elevated locomotion in voluntary wheel running test, which was suppressed by corticosterone synthesis inhibitor metyrapone [START_REF] Ebada | Corticosterone and dopamine D2/D3 receptors mediate the motivation for voluntary wheel running in C57BL/6J mice[END_REF]. In the same lines, higher basal corticosteroid may induce higher locomotion, particularly in females. A positive correlation between locomotion and serum corticosterone concentration was found in the current study confirming that an increase in serum corticosterone concentration may link with the higher locomotor activity. However, it was also observed that, male and female corticosterones were increased by 14.5% and 18.3% respectively, with decrease in locomotion by 13.1% in males and 8.7% in females after stress. This meant that CUMS significantly increased corticosterone concentration and reduced locomotion without sex differences.

Previous research reported higher corticosterone levels in female rats than male rats after CUMS [START_REF] Lu | Sex differences in the stress response in SD rats[END_REF]. The differences may be due to the duration, degree or the type of J o u r n a l P r e -p r o o f stress. With regards to the status of other depression-like behaviors, the present study found CUMS induced anhedonia and anxiety in both sexes without sex differences, which was in line with our previous findings [START_REF] Zhang | Minocycline ameliorates depressive behaviors and neuro-immune dysfunction induced by chronic unpredictable mild stress in the rat[END_REF][START_REF] Peng | EPA is More Effective than DHA to Improve Depression-Like Behavior, Glia Cell Dysfunction and Hippcampal Apoptosis Signaling in a Chronic Stress-Induced Rat Model of Depression[END_REF].

CUMS-induced changes in peripheral lymphocyte function

A chronic abnormal increase in corticosteroid concentration can desensitize glucocorticoid receptors located on macrophages/lymphocytes, which affects the release of pro-inflammatory factors and lymphocyte function [START_REF] Maes | The monocyte-T-lymphocyte hypothesis of major depression[END_REF][START_REF] Leonard | The immune system, depression and the action of antidepressants[END_REF][START_REF] Whalley | Glucocorticoid receptors and J o u r n a l P r e -p r o o f depression[END_REF]. Stress promotes the differentiation of naive CD4 T cells to Th1 and Th17 cells, and increases peripheral Th1/Th2 and IL-17/Treg cells ratio [START_REF] Zhao | TLR2 Involved in Naive CD4+ T Cells Rescues Stress-Induced Immune Suppression by Regulating Th1/Th2 and Th17[END_REF][START_REF] Hong | Imbalance between Th17 and Treg cells may play an important role in the development of chronic unpredictable mild stress-induced depression in mice[END_REF]. CD8 T cells also can promote the pro-inflammatory response in stressed mice [START_REF] Clark | CD8(+) T cells promote cytokine responses to stress[END_REF]. Increased pro-inflammatory IFN-γ/IL-4 (Th1/Th2) ratio and IL-17 (Th17) cytokines was found in stressed mice [START_REF] Hong | Imbalance between Th17 and Treg cells may play an important role in the development of chronic unpredictable mild stress-induced depression in mice[END_REF] and depressed patients [START_REF] Myint | Th1, Th2, and Th3 cytokine alterations in major depression[END_REF][START_REF] Ghosh | Circulating T helper 17 and IFN-γ positive Th17 cells in Major Depressive Disorder[END_REF]. Women show a longer and stronger immune/inflammatory response than men under an infection, tissue injury or stress [START_REF] Dragoş | The effect of stress on the defense systems[END_REF][START_REF] Pitychoutis | Of depression and immunity: does sex matter?[END_REF][START_REF] Bekhbat | Sex differences in the neuro-immune consequences of stress: Focus on depression and anxiety[END_REF]. In previous study, healthy women exhibited higher CD3 and CD4 T lymphocytes than healthy men in the blood [START_REF] Bouman | Gender difference in the non-specific and specific immune response in humans[END_REF]. Pro-inflammatory factors originating from T lymphocytes, such as IL-6, IL-12, IL-17, etc., are observed to be higher in females than males after inflammatory or stress stimulation [START_REF] Bekhbat | Sex differences in the neuro-immune consequences of stress: Focus on depression and anxiety[END_REF][START_REF] Cho | Sex bias in experimental immune-mediated, drug-induced liver injury in BALB/c mice: suggested roles for Tregs, estrogen, and IL-6[END_REF]. With regards to the sex differences in lymphocyte subtypes, the present study found lower CD8 but higher CD4 and ratio of CD4/CD8 in female than male rats in the control condition.

Interestingly, CUMS reversed this, by significantly increasing CD8 and decreasing CD4 and CD4/CD8 ratio in the blood of female rats. These sex differences remained with females having higher difference compared to males in the control group and when compared to control animals of the same sex. These may confirm that females have a stronger cell-mediated immunity dominated inflammatory response during infection or stress exposure [START_REF] Dragoş | The effect of stress on the defense systems[END_REF][START_REF] Miller | Depression and immunity: a role for T cells?[END_REF][START_REF] Beagley | Regulation of innate and adaptive immunity by the female sex hormones oestradiol and progesterone[END_REF]. Additionally, different studies reported no changes in blood CD4 and CD8 in female mice [START_REF] Silberman | Effects of chronic mild stress on lymphocyte proliferative response. Participation of serum thyroid hormones and corticosterone[END_REF], while decreased blood CD4 and CD4/CD8 ratio, but increased CD8 cells, as well as serum pro-inflammatory TNF-α, IL-6 and IFN-γ concentrations were found in male rats after CUMS [START_REF] Guan | Chronic unpredictable mild stress impairs erythrocyte immune function and changes T-lymphocyte subsets in a rat model of stress-induced depression[END_REF]. The reason for the variance between our observations and others may due to the CUMS paradigm used, or species differences. Importantly, our findings resemble clinical data that J o u r n a l P r e -p r o o f women have a stronger immune response than men in CD3 and CD4 lymphocytes and CD4/CD8 ratio in both healthy people [START_REF] Wikby | The immune risk profile is associated with age and gender: findings from three Swedish population studies of individuals 20-100 years of age[END_REF] and depressive patients [START_REF] Fagniart | Depression, gender and cellular immunity: influence of gender and severity of depression on the cellular immunity[END_REF].

To further explore the mechanism of CD4 dysfunction in depression, our study measured CD4 subtype CD25 Treg lymphocyte, where a decline of anti-inflammatory spleen CD25 Tregs after CUMS exposure was observed in both sexes. Although no positive correlation between sucrose preference and blood CD4/CD8 ratio was found in the present study, clinical investigations have reported a strong negative correlation between the Beck Depression Inventory (BDI) and percentages of CD3, CD4 and CD4/CD8 ratio [START_REF] Zdanowicz | Depression, family and cellular immunity: Influence of family relationships and cellular immunity on the severity of depression[END_REF]. The reason for the different findings may be due to the CUMS paradigm, species differences, sample number limit and different types of depression-like behavior (such as helplessness) tested. Moreover, a positive correlation between depression-like behavior (sucrose preference) and CD4 lymphocyte subtypes (spleen CD25) were found in the present study. This is in line with clinical data where patients diagnosed with major depression have lower blood CD25 Treg cells [START_REF] Li | Altered expression of CD4(+)CD25(+) regulatory T cells and its 5-HT(1a) receptor in patients with major depression disorder[END_REF]. Chronic stress significantly reduced CD25 Treg and induced depression-like behaviors, and these abnormal depression-like behaviors can also be found in CD25 Treg cell depleted mice [START_REF] Kim | CD4+CD25+ regulatory T cell depletion modulates anxiety and depression-like behaviors in mice[END_REF]. The reduction of blood CD25 Treg in major depression can be recovered by antidepressant, imipramine [START_REF] Li | Altered expression of CD4(+)CD25(+) regulatory T cells and its 5-HT(1a) receptor in patients with major depression disorder[END_REF][START_REF] Grosse | Circulating cytotoxic T cells and natural killer cells as potential predictors for antidepressant response in melancholic depression. Restoration of T regulatory cell populations after antidepressant therapy[END_REF].

Additionally, a positive correlation has been found between spleen CD25 and serum TGF-β1/IL-6 ratio in the present study. This is consistent with previous literature, where anti-inflammatory TGF-β can promote and anti-inflammatory IL-6 inhibits

Treg differentiation and production [START_REF] Bettelli | Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells[END_REF]. Translation of total T lymphocytes to Rag1 -/- mouse can significantly improve lipopolysaccharide induced inflammatory response and depression-like behaviors [START_REF] Laumet | Resolution of inflammation-induced depression requires T lymphocytes and endogenous brain interleukin-10 signaling[END_REF].

Overall, our data showed that females have a stronger basal immune background of CD4, CD8 lymphocytes and CD4/CD8 ratio. CUMS resulted in a larger decrease from basal CD4, CD4/CD8 ratio, but larger increase from basal CD8 in female than male rats. Although our study did not find sex differences in depression-like behaviors (anhedonia), the lymphocyte subtype change may still play an important role in a higher clinical risk of depression in women.

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CUMS-induced changes in peripheral cytokines

The monocyte-T-lymphocyte hypothesis of depression postulates that activated T lymphocyte subtypes-triggered inflammatory responses may induce the onset and development of depression [START_REF] Maes | The monocyte-T-lymphocyte hypothesis of major depression[END_REF][START_REF] Miller | Depression and immunity: a role for T cells?[END_REF]. With regards to the sex difference, the present study showed there was a higher serum IL-6 concentration in female than male rats in control situation. Increased plasma IL-6 concentration has been considered as a key factor for diagnosis of major depression, as well as the outcome of a therapeutic response [START_REF] Ting | Role of Interleukin-6 in Depressive Disorder[END_REF]. Higher IL-6 pro-inflammatory background may indicate increased depression risk in women. Even though there was an increase in serum IL-6 concentration in both sexes after CUMS exposure, the change within the same sex was not significant, which may be one reason why there was no sex differences in anhedonia. Other studies have also demonstrated that CUMS markedly increased serum and hippocampal IL-6 concentrations in male mice [START_REF] Liu | Anti-Depressant-Like Effect of Sinomenine on Chronic Unpredictable Mild Stress-Induced Depression in a Mouse Model[END_REF][START_REF] Shen | Avicularin Relieves Depressive-Like Behaviors Induced by Chronic Unpredictable Mild Stress in Mice[END_REF][START_REF] Zhao | Behavioral, inflammatory and neurochemical disturbances in LPS and UCMS-induced mouse models of depression[END_REF]. The IL-6 levels in the medial prefrontal cortex (mPFC) was elevated only in males but not in females after stress though females had higher basal IL-6 levels [START_REF] Jankovic | Inhibition of the fatty acid amide hydrolase changes behaviors and brain catecholamines in a sex-specific manner in rats exposed to chronic unpredictable stress[END_REF]. However, clinical studies have reported that women with major depression show higher plasma IL-6 concentration than men [START_REF] Qu | Sex difference in IL-6 modulation of cognition among Chinese individuals with major depressive disorder[END_REF], but no change in plasma IL-6 has also been observed in patients with major depression [START_REF] Euteneuer | Depression, cytokines and experimental pain: evidence for sex-related association patterns[END_REF]. This rise in plasma IL-6 concentration can be reversed by antidepressant paroxetine treatment in major depression, in both sexes [START_REF] Dong | Plasma levels of interleukin-6 and antidepressant response to Paroxetine in Chinese depressive patients[END_REF]. These varying findings may result from the severity and duration of the illness, or antidepressant treatment effects. Overall, although no sex differences were recorded after CUMS in our models, females having a higher basal IL-6 concentration might still play an important role in inflammation-related diseases. Present CUMS protocol (duration, degree or types of stress) and other depression-like behaviors (helplessness) should also be considered in the future studies, as gender differences of plasma IL-6 concentration have been found in patients with major depression [START_REF] Qu | Sex difference in IL-6 modulation of cognition among Chinese individuals with major depressive disorder[END_REF].

With regards to the status of other pro-and anti-inflammatory factors, the present study found that CUMS significantly decreased serum anti-inflammatory IL-10, but increased pro-inflammatory IFN-γ, IL-17 and IFN-γ/IL-10 ratio without sex differences. These findings are in line with our previous results that CUMS can J o u r n a l P r e -p r o o f damage the balance between pro-and anti-inflammatory factors, which were observed in depressed patients [START_REF] Zhang | Minocycline ameliorates depressive behaviors and neuro-immune dysfunction induced by chronic unpredictable mild stress in the rat[END_REF][START_REF] Peng | EPA is More Effective than DHA to Improve Depression-Like Behavior, Glia Cell Dysfunction and Hippcampal Apoptosis Signaling in a Chronic Stress-Induced Rat Model of Depression[END_REF].

CUMS-induced changes in hippocampal glia cells and neuroinflammation

In the central nervous system, CUMS exposure affected glia activity and the concentrations of cytokines in the hippocampus, as evidenced by decreased anti-inflammatory M2 factors IL-10 and GFAP, but increased pro-inflammatory M1 factors IFN-γ, IL-6, IL-17 and MI/M2 ratio IFN-γ/IL-10, without sex differences.

These findings are in line with our previous studies that CUMS induced imbalance between pro-/anti-inflammatory factors, and decreased astrocyte neuroprotective function, including suppression of the expression of GFAP and BDNF [START_REF] Zhang | Minocycline ameliorates depressive behaviors and neuro-immune dysfunction induced by chronic unpredictable mild stress in the rat[END_REF][START_REF] Peng | EPA is More Effective than DHA to Improve Depression-Like Behavior, Glia Cell Dysfunction and Hippcampal Apoptosis Signaling in a Chronic Stress-Induced Rat Model of Depression[END_REF].

Depression-like behaviors are significantly reversed by the anti-inflammatory drug minocycline through suppression of inflammatory responses both in males and females [START_REF] Zhang | Minocycline ameliorates depressive behaviors and neuro-immune dysfunction induced by chronic unpredictable mild stress in the rat[END_REF][START_REF] Wang | Microglial activation mediates chronic mild stress-induced depressive-and anxiety-like behavior in adult rats[END_REF]. These suggest that the balance between pro-and anti-inflammatory responses contribute to alleviate depression-like behaviors.

CUMS-induced changes in hippocampal neurotransmitter concentration

Numerous studies have demonstrated that activated microglia result in monoamine neurotransmitter deficiency by triggering inflammatory signals, releasing excess of pro-inflammatory factors, or decreasing BDNF through suppression of astrocyte functions, thereby causing depression [START_REF] Felger | Inflammatory cytokines in depression: neurobiological mechanisms and therapeutic implications[END_REF][START_REF] Beurel | The Bidirectional Relationship of Depression and Inflammation: Double Trouble[END_REF]. Indeed, the monoamine deficits have been extensively studied to explain various clinical phenotypes in psychiatric and neurologic diseases [START_REF] Hanin | Central neurotransmitter function and its behavioral correlates in man[END_REF][START_REF] Montgomery | Neurotransmitter system interactions revealed by drug-induced changes in motivated behavior[END_REF][START_REF] Eison | Serotonergic mechanisms in anxiety[END_REF]. Accordingly, most antidepressant treatments target NE, 5HT synthesis pathways and their receptors [START_REF] Schatzberg | Clinical efficacy of reboxetine in major depression[END_REF][START_REF] Tacke | Fluoxetine: an alternative to the tricyclics in the treatment of major depression?[END_REF]. With regards to the sex differences, the present study showed a lower basal hippocampal NE concentration in females when compared to male rats in control situation. As mentioned above, alterations in NE systems have been reported in depression and antidepressant drugs that increase NE levels are used in the treatment of depression [START_REF] Delgado | Depression: the case for a monoamine deficiency[END_REF][START_REF] Hamon | Monoamine neurocircuitry in depression and strategies for new treatments[END_REF]. In the present study, after CUMS exposure, a decrease in hippocampal NE concentration was found in both females and males, but statistically no significant sex differences were observed for this change in decreased percentage. This in line with our previous study that CUMS decreases NE concentrations in the hippocampus [START_REF] Peng | EPA is More Effective than DHA to Improve Depression-Like Behavior, Glia Cell Dysfunction and Hippcampal Apoptosis Signaling in a Chronic Stress-Induced Rat Model of Depression[END_REF].

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A previous study found lower levels of hippocampal NE in stressed female rats than stressed males [START_REF] Jankovic | Inhibition of the fatty acid amide hydrolase changes behaviors and brain catecholamines in a sex-specific manner in rats exposed to chronic unpredictable stress[END_REF]. Previous study has shown that serum norepinephrine decrease in female stressed mice was greater than that observed in male stressed mice, while the serum 5-HT drop in male stressed mice was greater than that in female mice [START_REF] Chen | Sex differences in peripheral monoamine transmitter and related hormone levels in chronic stress mice with a depression-like phenotype[END_REF]. The reason for the difference between our findings and others might due to the CUMS paradigm, the duration used, or age and species of the animal used. With regards to other neurotransmitters, in the present study, CUMS also decreased hippocampal 5-HT concentration without sex differences, which is in line with decreased hippocampal 5-HT and NE in our previous research [START_REF] Peng | EPA is More Effective than DHA to Improve Depression-Like Behavior, Glia Cell Dysfunction and Hippcampal Apoptosis Signaling in a Chronic Stress-Induced Rat Model of Depression[END_REF]. A significant positive correlation between sucrose preference and hippocampal neurotransmitters (NE and 5-HT) was also found, regardless of sex. These findings thus support the hypothesis that the deficiency of monoamine neurotransmitters induced depression-like behaviors after CUMS exposure, but likely does not explain sex difference in depression susceptibly.

Limitations and future directions

The data from the current study is not sufficient to provide clear evidence that stress could promote depressive-like behaviors in females when compared to males.

Future studies should try to standardize CUMS paradigm, keep view the duration and types of stress, animal species and age, as well as estrous cycle (females are more susceptible to stress and emotional). Moreover, other animal models of depression, such as olfactory bulbectomy, LPS or IL-1β inflammatory challenge model, social defeat, should be considered, because each model has its advantages and disadvantages. As well, helplessness (using forced swimming or tail suspension test), which is one of the core symptoms of depression, should be evaluated in the future.

Additionally, specific brain areas should be studied, such as prefrontal cortex, amygdala, and hypothalamus. The role of lymphocyte and inflammatory response in the CNS should further be investigated by using transgenic mice or rodents with related factor treatments in different sexes.

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Conclusion

The present study showed that CUMS significantly induced depression-like behaviors, which are related to the abnormalities in stress hormone, lymphocytes, inflammatory factors and monoamine neurotransmitters. More importantly, the present study found that several important parameters significantly differed in non-stressed females, such as higher locomotion, blood CD4/CD8 ratio, serum corticosterone and IL-6, but lower hippocampal NE concentration than male rats.

Even though CUMS failed to induce the sex differences, the original abnormalities in several depression-related factors may still play important role in female depression vulnerability. Nevertheless, the study results may be used as a reference to standardize depression models and also choose a suitable treatment for depression in different genders.

Highlights

1. CUMS induced depression-like behaviors, changed lymphocyte subtypes and inflammatory abnormalities in both males and females.

2. Females showed higher locomotion, blood CD4/CD8 ratio, serum corticosterone and IL-6, but lower hippocampal NE concentrations than males in control conditions.

3. Sex differences were observed in lymphocytes, both in control and stress conditions.

4. There was a positive correlation between sucrose preference and splenic CD25, and between sucrose preference and hippocampal neurotransmitters (NE and 5-HT), regardless of sex.

  Fig 1 summarizes the general study design. Briefly, stress group rats were subjected to CUMS for 49 days and control group rats no stress. During day 45-48, the sucrose preference test (SPT) was performed. During days 50-51 the open-field test (OFT) and elevated plus maze test (EPT) were conducted at 7:30-13:30. Rats were euthanized after one day's rest.

Fig1.

  Fig1. Timeline of the study CUMS: chronic unpredictable mild stress, PST: sucrose preference test, OFT: open-field test, EPT: elevated plus maze test, FCM: flow cytometry, WB: Western blots, HPLC: high performance liquid chromatography.

  the test, rats were acclimatized to the room for 1 h. After the test, the arena was cleaned with 0.5% EtOH. The number of open and closed arms entries and the time in open and closed arms, as well the number and time ratio of close/open, were J o u r n a l P r e -p r o o f quantified.

  into 500 μL heparin anticoagulant tubes (MB1908, Meilunbio, China) for flow cytometry. Serum samples were centrifuged at 1800 rpm for 10 min. Serum was collected and stored at -80°C. Following brain extraction, hippocampus were quickly dissected on ice, frozen in liquid nitrogen, and stored at -80℃ until whole hippocampus homogenization for subsequent ELISA and Western blot assays. The commercial rat ELISA kits were used to determine cytokines IL-6 (YT-0190R1), TGF-β1 (YT-0181R1) (Jiangsu Yu Tong Biotechnology Co., Ltd, China) and CORT (DG-70084D), IL-17 (DG-20148D), IFN-γ (DG-90958D), IL-10 (DG-70038D) (Beijing Dong Ge Biotechnology Co., Ltd, China) in both serum and hippocampal supernatant according to the manufacturer protocols.

  Medium (Tianjin Haoyang biological products Technology Co., Ltd, China). The single Lymphocyte suspension (2*10 6 /100μL) and 100 μL fresh anticoagulant blood J o u r n a l P r e -p r o o f was surface stained with CD3-APC (1920424, eBioscience), CD4-FITC (4321795, eBioscience), CD8-PerCP/Cy5.5 (1998249, eBioscience) and CD25-PE (4330019, eBioscience) for 30 min at room temperature in the dark. 2 ml erythrocyte lysis buffer

  pH of supernatants was adjusted to 3.8 with 1 M sodium acetate, and then supernatants were stored at -80℃ until use. For the HPLC analysis, 10 μL of the J o u r n a l P r e -p r o o f pH-adjusted supernatant was injected into an HPLC system with fluorescence detection (Agilent, Santa Clara, CA, USA). The neurotransmitters in the samples were separated by a C18 reverse-phase column (4.5*150 mm) (Agilent, Santa Clara, CA, USA) with a mobile phase containing sodium acetate and citric acid. The mobile phase was prepared as follows: 0.1 M sodium acetate was mixed 0.1 M citric acid in a 10:9 ratio, and adjusted to PH 3.5 (0.1 M sodium citric buffer), mixed with methanol in a ratio of 85:15 and then supplemented with sodium octane sulfonate (100 mg/L), Na 2 EDTA (5 mg/mL).2.11. Statistical analysisAll data were expressed as mean ± SEM and analyzed by SPSS 20.0 software (IBM SPSS Statistics software, SPSS Inc. Chicago). Parametric data were measured by two-way analysis of variance (ANOVA, factors CUMS (stress/control) and sex (male/female)). Non-parametric data were analyzed using Kruskal-Wallis test, followed by a Bonferroni-corrected post-hoc Wilcoxon-Mann-Whitney U-test for significant data. Significance was set at P<0.05 in all tests. A non-parametric Spearman rank correlation test was used to analyze linear correlations.

Fig 2 .

 2 Fig 2. CUMS decreased weight and sucrose consumption in both sexes. A) Sucrose preference. B) Body weight over seven weeks. All data are expressed as the mean ± SEM (N=10 per group). ** P<0.01 CUMS main effect; post-hoc test: # P<0.05 CUMS vs. control.

Fig 3 .

 3 Fig 3. The different behavioral changes of male and female rats in the open field test. A) The locomotion in the open field maze. B) The number of entries into the central zone. C) The time spent in the central zone. On the left is the track of rats in the open field box. All value expressed as the mean ± SEM (N=10 per group). * P<0.05, ** P<0.01 CUMS main effect, && P<0.05, && P<0.01 sex main effect.

  was a significant main effect of CUMS on the time in the open arm (F 1,36 =7.662, P<0.01), the ratio of entries in open over closed arms(F 1,36 =9.202, P<0.01) and the ratio of time spent in the open over closed arms (F 1,36 =8.155, P <0.01) (Fig. 4B, C, D). CUMS significantly decreased the time spent in the open arms, the number of entries into the open arm (H=11.842, P<0.01) and the ratio of open to closed arm entries in both males and females. J o u r n a l P r e -p r o o f

Fig 4 .

 4 Fig 4. The anxiety-like behaviors of male and female rats in the elevated plus maze. A) The number of entries into the open and close arms. B) The time spent in the open and close arms. C) The ratio of open to closed arm entries. D) The ratio of time spent in the open over the closed arm. On the top is the track of rats in the elevated plus maze. All values are expressed as the mean ± SEM (N=10 per group). ** P<0.01 CUMS main effect; post-hoc test: # P<0.05 CUMS vs. control.

Fig 5 .

 5 Fig 5. CUMS increased serum CORT concentration both in females and males. A) Serum CORT concentration. B) Positive correlation between locomotion and serum CORT, sexes combined. All data are expressed as the mean ± SEM (N=8 per group). post-hoc test: & P<0.05, && P<0.01 females vs. males, # P<0.05 CUMS vs. control.

Fig 6 .

 6 Fig 6. Male and female lymphocyte subtypes in blood and spleen after CUMS. A) The percentage of CD4 and CD8 in spleen. B) The percentage of CD4 and CD8 in blood. C) The percentage of CD25 in spleen and blood. D) The ratio of CD4 and CD8 in spleen and blood. E) Correlation between sucrose preference and blood CD4/8 ratio, F) Correlation between spleen CD25 and sucrose preference, sexes combined. All data are expressed as the mean ± SEM (N=6 per group). ** P<0.01 CUMS main effect; post-hoc test: ## P<0.01 CUMS vs. control; & P<0.05

Fig 7 .

 7 Fig 7. Serum cytokines after CUMS in male and female rats. A) IL-10 and IFN-γ levels. B) IL-6 and TGF-β1 levels. C) The ratio of IFN-γ/IL-10. D) The ratio of TGF-β1 IL-6. E) IL-17. F) Positive correlation between spleen CD25 levels and serum TGF-β1/IL-6 ratio, sexes combined. All data are expressed as the mean ± SE (N=8 per group). * P<0.05, ** P<0.01 CUMS main effect, && P<0.01 sex main effect; post-hoc test: ## P<0.01 CUMS vs. control.

Fig 8 . 3 . 8 .

 838 Fig 8. Hippocampal glia cell expression after CUMS in male and female rats. A) CD11b expression in the hippocampus. B) GFAP and BDNF expression in the hippocampus. All values are expressed as the mean ± SEM (N=6 per group). * P<0.05, ** P<0.01 CUMS vs. control. ** P<0.01 CUMS main effect; post-hoc test: # P<0.05 CUMS vs. control.

Fig 9 .

 9 Fig 9. Hippocampal cytokines after CUMS in male and female rats. A) IL-10 and IFN-γ. B) IL-6 and TGF-β1. C) The ratio of IFN-γ/IL-10. D) IL-17. All values are expressed as the mean ± SEM (N=8 per group). ** P<0.01 CUMS main effect; post-hoc test: ## P<0.01 CUMS vs. control.

Fig 10 .

 10 Fig 10. Concentration of NE and 5HT in the hippocampus of male and female rats. A) Hippocampal NE concentration. B) Hippocampal 5-HT concentration. C) Correlation between hippocampal NE and sucrose preference, sexes combined. D) Correlation between hippocampal 5-HT and sucrose preference, sexes combined. All data are expressed as the mean ± SEM (N=6 per group). ** P<0.01 CUMS main effect; && P<0.01 sex main effect.

Fig11.

  Fig11. Potential schematic and result's summary of the study. CUMS: chronic unpredictable mild stress, HPA: hypothalamic pituitary adrenocortical axis. "↑" represent increase, "↓" represent decrease. "≈"represent similar increased or decreased percentage when compared to control animals of the same sex. ">"represent larger increased or decreased percentage when compared to control animals of the same sex.

Table 1 .

 1 Summary of the CUMS protocol

	J o u r n a l P r e -p r o o f
	Stressors	Experimental days
	Cold exposure at 4 °C for 1 h	2, 8, 22, 30, 37, 44, 49
	Swim stress at 18 °C for 10 min	3, 10, 20, 26, 34, 41, 46
	Restraint for 1 h	1, 9, 19, 27, 33, 45, 49

Females showed higher locomotion, blood CD4/CD8 ratio, serum corticosterone and IL-6, but lower hippocampal NE concentrations than males in control conditions.

Sex differences were observed in lymphocytes, both in control and stress conditions.

There was a positive correlation between sucrose preference and splenic CD25, and between sucrose preference and hippocampal neurotransmitters (NE and 5-HT), regardless of sex.J o u r n a l P r e -p r o o f

Acknowledgments

This work was supported by grants to CS from the Natural Science Fund of Guangdong Province (2021A1515011579), Shenzhen Science and Technology Plan (International Cooperation Research) Project (GJHZ20190823111414825), Zhanjiang Science and Technology Project (2021A05046), Guangdong Science and Technology Project (2016B020235001).

Author contributions

CS designed the experiments and edited the manuscript. CZ and BPL performed the experiments and analyzed the data. CZ drafted the paper. JLP, HS and UKK modified the paper and assisted with statistical analysis. All authors have read, discussed, and approved the final version of the manuscript.

J o u r n a l P r e -p r o o f

Conflict of interest

The authors declare no conflicts of interest.


Mathieu Levaillant

Charlotte Loury

Aurélien Venara

Jean-François Hamel-Broza

Guillaume Legendre

Response: Is there still an indication for episiotomy? Results from a French national database analysis

Response: Is there still an indication for episiotomy? Results from a French national database analysis Mathieu Levaillant1 2 , Charlotte Loury 3 4 , Aurélien Venara 3 5 , Jean-François Hamel-Broza 2 4 , Guillaume Legendre We fully agree with Matthieu Dap and his colleagues that the risk of OASIS should be modulated according to the type of instrument used: spatulas, forceps, or vacuum cups. From the same perspective, it is likely that vacuum cups should be preferred to limit the risk of OASIS. However, the message of our study, based on French medico-administrative data covering the whole population, was intended to be more general, proposing a more global vision on the indications of episiotomy. Indeed, it appears that episiotomy seems to be able to preserve certain indications in the instrumental delivery of primiparous women, independently of the type of extraction carried out. Unfortunately, the databases do not allow this level of precision to be obtained in a reliable manner.

Overall, the risk of OASIS may be lower with the use of vacuum cups rather than forceps, and forceps remains associated with a higher rate of OASIS. For example, in the meta-analysis by Lund et al. in 2016, medio-lateral episiotomy or lateral episiotomy significantly reduces the risk of OASIS even in vacuum-assisted deliveries in primiparous women (OR 0.53, 95% CI 0.37-0.77).1 Furthermore, the work of Levin et al. in 2021 found an identical rate of OASIS in vacuum delivery as in spontaneous vaginal delivery, despite a higher rate of episiotomy in the vacuum group.2 Our results, even if they remain cautious regarding the role of episiotomy in cases of instrumental delivery in primiparous women, thus seem justified to us. These results are consistent with the review of the literature by Sultan et al. on the role of right lateral episiotomy in operative delivery.3 Prospective studies (some of which are in progress in France) or well-conducted randomized trials should make it possible to better specify the risk of OASIS according to each instrument and the place of preventive medio-lateral episiotomy in this population of nulliparous women considered at risk.

  University of Angers, CHU Angers, University of Rennes, Inserm, EHESP, Irset (Institut de Recherche en Santé, Environnement et Travail), Angers, France.

	3 6
	1 -University of Lille, CHU Lille, ULR 2694 -METRICS: Évaluation des Technologies de Santé et des
	Pratiques Médicales, Lille, France.
	2 -Methodology and Biostatistics Department, Angers University Hospital, University of Angers,
	Angers, France.
	3 -Faculté de Santé, Département de Médecine, Angers, France.
	4 -Service de Gynécologie-Obstétrique, CHU d'Angers, Angers, France.
	5 -Service de Chirurgie Digestive, CHU d'Angers, Angers, France.
	6 -UMR_S1085,

-Lund NS, Persson LKG, Jangö H, Gommesen D, Westergaard HB. Episiotomy in vacuum-assisted delivery affects the risk of obstetric anal sphincter injury: a systematic review and meta-analysis. Eur J Obstet Gynecol Reprod Biol.

2016; 207: 193-199. 2 -Levin G, Friedman T, Shai D, Alcalay M, Ram E, Meyer R. The association between vacuumassisted vaginal delivery and anal sphincter injury. Int Urogynecol J 2021; 32(7): 1771-7, 1777.