Kahina Boukari

Géri Meduri

Sylvie Brailly-Tabard

Jean Guibourdenche

Maria-Luisa Luisa Ciampi

Nathalie Massin

Laetitia Martinerie

Jean-Yves Picard

Rodolfo Rey

Marc Lombès

Geri Meduri

MD Jacques Young
email: jacques.young@bct.aphp.fr

Lack of Androgen Receptor Expression in Sertoli Cells accounts for the Absence of Anti-Mullerian Hormone Repression during Early Human Testis Development

Keywords: Human testis, Fetal, Newborn, Anti-Mullerian Hormone, Androgen receptor, Sertoli cell, Follicle-stimulating-hormone receptor, congenital hypogonadotropic hypogonadism

Objective: We postulated that a lack of androgen receptor (AR) expression in Sertoli cells (SC) might explain why these processes do not occur during early human development.

Methods and patients: Using immunohistochemistry and quantitative PCR we examined the relationship between AR, AMH and FSH receptor (FSHR) expression in fetal, newborn and adult human testis. The ability of T to repress AMH secretion was evaluated in male newborns, neonates and two adults with androgen insensitivity syndrome (AIS) and also in vitro using SMAT1 Sertoli cells.

Results: FSHR was present in SC at all developmental stages. In fetal and newborn testis, AR was expressed in peritubular and Leydig cells but not in SC. This coincided with the absence of spermatogenesis and with strong SC AMH expression. In adult testis, spermatogenesis was associated with AR expression and with a decrease in SC AMH content. Accordingly, AR mRNA expression was lower and AMH mRNA expression higher in fetal testes than in adult testes. In AIS patients combined gonadotropin stimulation induced an increase in circulating T and AMH, a finding consistent with a failure of TT to repress AMH in the absence of AR signalling. Finally, direct androgen repression of AMH only occurred in ARexpressing SMAT1 cells.

INTRODUCTION

In the human adult testis, spermatogenesis is under the control of two gonadotropins, namely follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH acts directly on Sertoli cells (SC), while LH induces testosterone (T) production after Leydig cell stimulation. Intratesticular testosterone (TT) acts via a paracrine mechanism on androgen receptors (AR) expressed by target cells situated in the seminiferous tubules. In adulthood, the action of androgens on the seminiferous tubules is essential for full, quantitatively normal spermatogenesis and fertility. Most evidence suggests that this effect is mediated through an effect on SC, although the precise mechanisms are unclear. Recently, spermatogenesis was found to be arrested in mice specifically lacking AR in their SC [START_REF] Chang | Infertility with defective spermatogenesis and hypotestosteronemia in male mice lacking the androgen receptor in Sertoli cells[END_REF][START_REF] De Gendt | A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis[END_REF][START_REF] Holdcraft | Androgen receptor function is required in Sertoli cells for the terminal differentiation of haploid spermatids[END_REF]. Anti-Mullerian hormone (AMH) is produced by SC from fetal life until puberty and is responsible for Mullerian duct regression in male fetuses. AMH and T serum levels correlate negatively during puberty and adulthood, indicating that TT is responsible for inhibiting SC AMH production. This was confirmed in adult congenital hypogonadotropic hypogonadism (CHH) patients, whose testicular AMH secretion is inhibited by both hCG and androgen administration [START_REF] Young | Testicular anti-mullerian hormone secretion is stimulated by recombinant human FSH in patients with congenital hypogonadotropic hypogonadism[END_REF][START_REF] Young | Antimullerian hormone in patients with hypogonadotropic hypogonadism[END_REF]. AMH and T levels are both high during fetal and neonatal life [START_REF] Rey | Anti-mullerian hormone and testosterone serum levels are inversely during normal and precocious pubertal development[END_REF][START_REF] Meyts | Expression of anti-Mullerian hormone during normal and pathological gonadal development: association with differentiation of Sertoli and granulosa cells[END_REF], indicating that testicular T is unable to repress AMH during these periods. One important physiological issue is why the human testis is unable to produce sperm and to repress AMH secretion during fetal and neonatal life, despite stimulation by chorionic and pituitary gonadotropins, respectively. The aim of this study was to test the hypothesis that the absence of spermatogenesis and the AMH repression observed during human fetal and newborn life are related to the absence of AR in SC, despite FSH receptor (FSHR) expression. For this purpose, we compared the expression profiles of AR, FSHR and AMH proteins and their messenger RNAs in human fetal, newborn and adult testes by means of immunohistochemistry and real-time quantitative PCR (qPCR). In addition, we compared T and AMH levels in umbilical arterial cord blood of male newborns, 20to 30-day-old neonates, and adults with normal or altered androgen sensitivity, in order to evaluate the ability of T to repress AMH secretion. Our results clearly demonstrate that in humans, AR expression in Sertoli cells is required both to induce spermatogenesis and to repress AMH.

MATERIALS AND METHODS

Tissues collection and quality

Archival paraffin-embedded human testicular samples were collected at different stages of development. Archival fetal testis samples (n=46), collected at 14 to 35 weeks of gestation, were selected from the organ bank of the Pathology Institute of Bari University with local ethics committee approval. Newborn (n=10) and adult (n=10) human testis samples were collected between 2 days and 6 months and between 20-40 years of age, respectively. These samples were obtained from the licensed collections of three pathology departments (Bicêtre, Créteil Intercommunal and Necker-Enfants Malades Hospitals, Paris). Snap-frozen testis specimens from 9 human fetuses of 16 to 38 weeks and from adult men were also obtained from the same licensed tissue collection (French Bioethics law n° 2004-800). The fetal samples originated from fetuses that had died in utero and had been rapidly delivered and autopsied within 24-36 h postmortem. None of the terminations were performed for fetal abnormalities. Tissue integrity was demonstrated as described in [START_REF] Boukari | Human fetal testis: source of estrogen and target of estrogen action[END_REF]. Neonatal testis samples were obtained from cases of sudden infant death, and adult samples were obtained from men undergoing surgical investigation for obstructive azoospermia. The selected samples had a normal karyotype (46 XY) and normal macroscopic and histological features.

Immunohistochemistry

Briefly, 5-µm-thick tissue sections were deparaffinized and rehydrated in successive baths of toluene and graded alcohol solutions. Slides were then subjected to microwave antigen retrieval for 15 min in pH 6 citrate buffer. For FSHR immunostaining the sections were preincubated with ready-to-use proteinase K (DakoCytomation, Carpinteria, CA) for 10 min at room temperature prior to 15 min of microwaving at full power in Tris-EDTA buffer pH 9. Slides were incubated overnight at 4°C with the following primary antibodies: anti-3-βhydroxysteroid-dehydrogenase (3 βHSD) (kind gift from Prof. Van-Luu The, Laval University, Canada), anti-AMH (9), anti-FSH receptor (FSHR 323) [START_REF] Vannier | Anti-human FSH receptor monoclonal antibodies: immunochemical and immunocytochemical characterization of the receptor[END_REF] and antiandrogen receptor (sc-816, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:2000, 1:1000 and 1:50 dilution, respectively. Bound immunoglobulins were revealed with a streptavidin-biotinperoxidase-aminoethylcarbazole kit (LSAB+, DakoCytomation).

RT-PCR

Total RNA was isolated from frozen samples with the TRIZOL reagent (Invitrogen, Cergy Pontoise, France) as recommended by the manufacturer. RNA integrity was checked on agarose gel before processing for RT-PCR as previously described [START_REF] Boukari | Human fetal testis: source of estrogen and target of estrogen action[END_REF] Real-time RT-PCR Specific AMH and AR gene expression was quantified by real-time PCR (see Supplemental Table 1 for primers). Total RNA, extracted as described above, was retrotranscribed and amplified on an ABI 7300 Sequence Detector (Applied Biosystems, Foster City, CA) as previously described [START_REF] Boukari | Human fetal testis: source of estrogen and target of estrogen action[END_REF]. Ribosomal 18S RNA was used as the internal control for data normalization. Results are means ± SEM of at least two independent analyses of at least two different reverse-transcribed samples. The relative expression level of each gene is expressed relative to 18S RNA (attomoles of specific gene per femtomole of 18S).

T and AMH measurements in umbilical cords of male newborns, neonates and adults Neonatal blood samples were obtained from umbilical cords arteries of 17 fullterm eutrophic male newborns immediately after delivery. Written informed consent was obtained from the mothers. The study was conducted in accordance with the Declaration of Helsinki and after approval from the local ethics committee. Blood samples were also obtained between day 20 and 30 of life from 21 normal boys with intrascrotal testes, and from 20 normal fertile males aged 17-37 years being evaluated in the context of female partner infertility. This part of the study was also approved by the local ethics committee, and we only used surplus serum taken for diagnostic purposes. Plasma testosterone (T) was measured with a commercial RIA method with a detection limit of 0.06 ng/ml (0.19 nmol/l) and intra-and interassay variation coefficients of 5.8% and 8.0%, respectively. Serum AMH levels were measured with an ELISA method (AMH/Mullerian-inhibiting substance ELISA, Immunotech-Beckman, Marseille, France), as previously described (4). The detection limit was 0.7 pmol/L (0.1 ng/ml), and the intra-and interassay coefficients of variation were 5.3% and 8.7%, respectively, for a serum AMH concentration of 35 pmol/l and 4.9% and 7.8% for a serum AMH concentration of 1100 pmol/l.

Clinical investigation of men with moderate androgen insensitivity syndrome (MAIS) or congenital hypogonadotropic hypogonadism (CHH)

We analyzed testicular responses to combined human gonadotropins in two azoospermic MAIS patients by comparison with 10 CHH patients receiving gonadotropin combination therapy with hCG (Organon, Puteaux, France) and rhFSH (recombinant human FSH) (Gonal-F®, Serono, Aubonne, Switzeland) at respective doses of 1500 IU and 150 IU, three times a week, for infertility. MAIS patient #1, who was hemizygous for the M780I mutation of the AR gene, has been described in detail elsewhere [START_REF] Rodien | Different phenotypes in a family with androgen insensitivity caused by the same M780I point mutation in the androgen receptor gene[END_REF]. MAIS patient #2, a Caucasian boy, was first seen at 19 years of age with bilateral gynecomastia and impaired virilization but no hypospadias. Testicular volume was 10 ml bilaterally (normal range 15-30 ml), the penis was less than 4 cm long and his pubic hair was at Tanner stage III. His total plasma T level was 13.4 ng/mL (normal, 3.2-9.7 ng/mL, x 3.467 = nmol/L), and his LH was 14.8 IU/L (normal, 4-7 IU/L). Analysis of two semen samples showed azoospermia. The previously reported A to G transition [START_REF] Shkolny | Discordant measures of androgen-binding kinetics in two mutant androgen receptors causing mild or partial androgen insensitivity, respectively[END_REF] was found in exon 8 at nucleotide position 2973 causing an unconserved arginine residue to be replaced by a glycine (R871G). Ten previously untreated men, aged 18-31 years, with idiopathic CHH (n=5) or Kallmann syndrome (n=5), were selected for diagnosis and choice of therapy. The diagnostic criteria for CHH were as reported elsewhere [START_REF] Young | Testicular anti-mullerian hormone secretion is stimulated by recombinant human FSH in patients with congenital hypogonadotropic hypogonadism[END_REF][START_REF] Young | Antimullerian hormone in patients with hypogonadotropic hypogonadism[END_REF][START_REF] Nistal | Morphological and histometric study on the human Sertoli cell from birth to the onset of puberty[END_REF][START_REF] Salenave | Kallmann's syndrome: a comparison of the reproductive phenotypes in men carrying KAL1 and FGFR1/KAL2 mutations[END_REF]. None of the patients had a history of cryptorchidism. All had low circulating T and gonadotropin levels. None had previously received gonadotropin or androgen replacement therapy. The patients were offered treatment consisting of FSH combined with hCG in order to increase testicular size, virilization and fertility as previously reported [START_REF] Young | Testicular anti-mullerian hormone secretion is stimulated by recombinant human FSH in patients with congenital hypogonadotropic hypogonadism[END_REF][START_REF] Young | Antimullerian hormone in patients with hypogonadotropic hypogonadism[END_REF][START_REF] Nistal | Morphological and histometric study on the human Sertoli cell from birth to the onset of puberty[END_REF]. All the subjects gave their informed written consent to participate in the study, which was approved by the local ethics committee. Treatment was always well tolerated and compliance, evaluated by measuring circulating FSH and T levels, was excellent (see Results). AMH was assayed in blood to evaluate the testicular response. In MAIS and CHH patients, blood samples were drawn before treatment and every month during combined treatment; hormone measurements were performed in a single assay.

Cells culture and transfection SMAT1 cells were cultured as described in [START_REF] Lukas-Croisier | Follicle-stimulating hormone increases testicular Anti-Mullerian hormone (AMH) production through sertoli cell proliferation and a nonclassical cyclic adenosine 5'monophosphate-mediated activation of the AMH Gene[END_REF]. Cells were seeded at a density of 2 x 10 5 /well in six-well plates and transiently transfected with 1 µg of human AR expression vector (hAR-pcDNA3) using the Lipofectamine reagent (Invitrogen, Carlsbad, CA92008). In brief, one day after initial plating, DMEM with 10% fetal calf serum was replaced by DMEM with Dextran-coated charcoal treated serum., Transfections were performed 24 h later. Fresh medium containing 10 -5 M forskolin alone or combined with 10 -7 M dihydrotestosterone was added to the cells for 24 h. The cells were then rinsed twice with PBS and processed for RNA extraction and real-time PCR as described above.

Western blot analysis and immunoprecipitation assay

Total protein extracts were prepared from SMAT1 cells transiently transfected or not with hAR-pcDNA3. Immunoprecipitation was performed by incubating 0.5 mg of total proteins with 1 µg of anti-AR antibody (sc-816, Santa-Cruz) overnight at 4°C. Immunoprecipitates were purified with protein A Sepharose CL-4B beads, and submitted to Western blot analysis using the same antibody at 1:200 dilution, followed by incubation with a peroxidase-conjugated goat anti-rabbit antibody (1:15000, Vector Laboratories, Burlingame, CA). The blots were visualized with the ECL+ detection kit (GE Healthcare).

RESULTS 3β β β βHSD expression in human testis

We first examined the immunoexpression pattern of the key enzyme 3βHSD in order to evaluate the steroidogenic activity of Leydig cells. As expected, the protein was located in the cytoplasm of Leydig cells in all fetal (Fig. 1A), newborn (Fig. 1B) and adult (Fig. 1C) testicular samples.

Immunohistochemical detection of AR and AMH in human testis

To determine the time course of AR and AMH expression during human testicular development, which might account for the relative testosterone resistance of SC, we compared AR and AMH expression in fetal, newborn and adult testes. In fetal and newborn testes, AR protein was detected in Leydig and myoid peritubular cells, but not in germ cells or SC (Fig. 1D and1E). The absence of AR in SC coincided with very strong AMH expression in the same cells of all fetal and neonatal testis samples (Fig. 1G and1H). In contrast, in all adult human testis samples examined, AR was detected not only in Leydig cells and myoid peritubular cells but also in SC (Fig. 1F), whereas AMH was not expressed (Fig. 1I). Semi-quantitative results for AR and AMH expression during human testis development are summarized in Table 1.

AR and AMH mRNA levels in human fetal testis

To examine quantitative changes in AMH and AR expression during testis development, mRNA levels of these genes were determined by real-time quantitative PCR in 10 fetal testes obtained at 16 (n= 2), 20 (n=1), 21 (n=1), 22 (n=1), 24 (n=2), 25 (n=2) and 38 (n=1) weeks of gestation, by comparison with adult testes (Fig. 2).

The relative expression level of AR mRNA in fetal testis was 30% or lower than that in adult testis (Fig. 2A). This difference was probably due to the absence of AR expression in fetal testis SC. We then examined AMH mRNA expression and found that levels were 2-to 30-fold higher in fetal than in adult testes (Fig. 2B). Thus, the AR/AMH mRNA ratio, an index of AR expression in SC, was 151 ± 8 in adult testis, compared to 1-20 in fetal testis, in keeping with the lack of AR immunoexpression in fetal SC.

FSHR expression in human testis

To determine whether the lack of spermatogenesis in the fetal and newborn testes is related to the lack of FSHR during these periods of life, we studied FSHR expression by immunohistochemistry and RT-PCR. As shown in Fig. 3A-C, FSHR immunoreactivity was localized to the SC membrane in fetal, newborn and adult testis. Furthermore, we confirmed that, as in adult testis, FSHR mRNA is also detected in human fetal testis (Fig. 3D). Using FSHR as another SC marker, we confirmed that the AR/FSHR mRNA ratio is higher in adult testis (406 ± 25) than in fetal testis (7.5 ± 1.5), providing additional support for the lack of AR expression in SC during human fetal development.

Inability of combined gonadotropin stimulation to repress AMH in adult patients with an AR signaling defect

We then compared the testicular responses to combined hCG and rhFSH in two adult MAIS patients and 10 CHH patients used as positive controls of preserved AR signaling in post-pubertal testes (Fig. 4A left panel). As expected (4, 5) gonadotropin therapy increased the serum levels of FSH (from 0.6 ± 0.3 to 11.2 ± 2.2 IU/L) and T (from 0.46 ± 0.2 to 5.8 ± 2.2 ng/ml) in CHH patients leading to a concomitant collapse of AMH levels (from 406 ± 274 to 37 ± 18 pmol/L; P<0.001) which reached normal adult levels (Fig. 4A, right panel). In contrast, in MAIS patients, despite a further increase in their plasma T levels induced by gonadotropin administration (from 11-13.4 to 15-19.4 ng/ml), the serum AMH level also rose, clearly demonstrating that, in these adult patients, testicular T is unable to repress AMH secretion in the absence of functional AR and therefore to prevent the AMH stimulation induced by rhFSH. This response pattern was similar to that observed during the prenatal and postnatal period in which, despite relatively high circulating T levels driven by chorionic and pituitary gonadotrophin stimulation, mean AMH levels remain significantly higher than in post-pubertal men (6) (Fig. 4A, right panel). Interestingly, AMH levels were significantly higher in 20-to 30-dayold boys than in newborns, a finding consistent with our demonstration of FSHR expression in SC and its stimulation by FSH during the early post-natal period [START_REF] Bergada | Time course of the serum gonadotropin surge, inhibins, and anti-Mullerian hormone in normal newborn males during the first month of life[END_REF]. In addition, strong AMH immunostaining was observed in SC of both adult patients with AIS and untreated CHH patients (Fig. 4B), contrasting with undetectable AMH expression in adults with normal testicular T and androgen sensitivity (see Fig. 1 panel I). Collectively, our results clearly indicate that repression of AMH secretion requires both an appropriate intratesticular T levels and functional AR signalling in SC.

AMH repression by androgens requires functional androgen receptors in SMAT1 cells

To investigate direct androgen regulation of AMH in a Sertoli cell model, we examined endogenous AMH expression by means of real-time PCR in SMAT1 cells, an immortalized immature Sertoli cell line which does not express AR (Fig. 5A). Forskolin induced a 2.5-fold increase in AMH expression (Fig. 5B) through a PKAdependent mechanism, as already reported [START_REF] Lukas-Croisier | Follicle-stimulating hormone increases testicular Anti-Mullerian hormone (AMH) production through sertoli cell proliferation and a nonclassical cyclic adenosine 5'monophosphate-mediated activation of the AMH Gene[END_REF]. However, coadministration of dihydrotestosterone did not affect forskolin-induced AMH expression. In contrast, when SMAT1 cells were transiently transfected with a human AR expression vector (Fig. 5A andC), forskolin-stimulated AMH mRNA levels returned to basal values after dihydrotestosterone exposure, providing the first evidence that functional AR are crucial for full AMH repression by androgens in Sertoli cells.

DISCUSSION

The role of androgen receptor (AR) in spermatogenesis has been the subject of intense interest for many years. Multiple rodent studies of androgen withdrawal and disruption of AR activity by surgical, chemical or genetic means have produced convergent results: spermatogenesis rarely occurs in the absence of androgens and functional AR. It has been assumed that the main cellular mediator of this regulatory function is the Sertoli cell (SC), given its intimate contact with germ cells devoid of AR [START_REF] Suarez-Quian | Androgen receptor distribution in adult human testis[END_REF][START_REF] Bremner | Immunohistochemical localization of androgen receptors in the rat testis: evidence for stage-dependent expression and regulation by androgens[END_REF][START_REF] Berensztein | Expression of aromatase, estrogen receptor alpha and beta, androgen receptor, and cytochrome P-450scc in the human early prepubertal testis[END_REF]. Our findings endorse this assumption. Other rodent studies have revealed the major role played by SC AR in the completion of spermatogenesis [START_REF] Johnston | Murine germ cells do not require functional androgen receptors to complete spermatogenesis following spermatogonial stem cell transplantation[END_REF]. Several groups (1-3) recently generated mice with SC-selective AR knockout (SCARKO mice), opening up new possibilities to elucidate the respective contributions to spermatogenesis of AR expression in different testicular cell types. Thus, despite normal testicular descent, all SCARKO mice generated to date display arrested spermatogenesis. These results prove that the initiation and maintenance of spermatogenesis are both crucially dependent on AR activation in SC. Several authors, while studying the profile of AR expression in SC during post-natal development of normal rodents and non human primates [START_REF] Al-Attar | Hormonal and cellular regulation of Sertoli cell anti-Mullerian hormone production in the postnatal mouse[END_REF][START_REF] Sharpe | Proliferation and functional maturation of Sertoli cells, and their relevance to disorders of testis function in adulthood[END_REF][START_REF] Mckinnell | Comparison of androgen receptor and oestrogen receptor beta immunoexpression in the testes of the common marmoset (Callithrix jacchus) from birth to adulthood: low androgen receptor immunoexpression in Sertoli cells during the neonatal increase in testosterone concentrations[END_REF][START_REF] Majdic | Immunolocalisation of androgen receptor to interstitial cells in fetal rat testes and to mesenchymal and epithelial cells of associated ducts[END_REF], failed to detect immunochemical AR expression in the immediate post-natal period. This could explain why the elevated T levels present in rodent and marmoset testis are unable to induce morphological maturation of SC, or to trigger and maintain spermatogenesis, despite high levels of androgens and gonadotropins in the fetus and neonate. The main purpose of the present study was to extend to humans this concept of a physiological state of transient androgen insensitivity of SC during fetal and neonatal life. Our results clearly show that, as reported for rodents and marmoset, AR protein is present in Leydig and peritubular cells of fetal and neonatal human testis, but not in SC. This expression pattern is in keeping with the absence of spermatogenesis, as expected [START_REF] Chemes | Physiologic Androgen Insensitivity of the Fetal, Neonatal, and Early Infantile Testis Is Explained by the Ontogeny of the Androgen Receptor Expression in Sertoli Cells[END_REF](26)[START_REF] Muller | Quantification of germ cells and seminiferous tubules by stereological examination of testicles from 50 boys who suffered from sudden death[END_REF], in our samples. During these two periods of human testicular development, hCG-and LH-induced biosynthesis of T is activated, as indicated by the strong 3βHSD expression in Leydig cells. This lack of AR expression in SC during early testicular development contrasted with the expected [START_REF] Suarez-Quian | Androgen receptor distribution in adult human testis[END_REF] strong AR expression we observed in these cells in sperm-producing adult human testis. The role of FSH in the initiation of human spermatogenesis remains controversial [START_REF] Lukas-Croisier | Follicle-stimulating hormone increases testicular Anti-Mullerian hormone (AMH) production through sertoli cell proliferation and a nonclassical cyclic adenosine 5'monophosphate-mediated activation of the AMH Gene[END_REF][START_REF] Lindstedt | Follitropin (FSH) deficiency in an infertile male due to FSHbeta gene mutation. A syndrome of normal puberty and virilization but underdeveloped testicles with azoospermia, low FSH but high lutropin and normal serum testosterone concentrations[END_REF][START_REF] Phillip | Male hypogonadism due to a mutation in the gene for the beta-subunit of follicle-stimulating hormone[END_REF][START_REF] Tapanainen | Men homozygous for an inactivating mutation of the follicle-stimulating hormone (FSH) receptor gene present variable suppression of spermatogenesis and fertility[END_REF][START_REF] Plant | The functional significance of FSH in spermatogenesis and the control of its secretion in male primates[END_REF][START_REF] Layman | FSH beta gene mutations in a female with partial breast development and a male sibling with normal puberty and azoospermia[END_REF]. In order to confirm that the lack of FSH receptor expression is not responsible for the lack of spermatogenic development in the fetal and early post-natal testis, we examined the FSHR expression pattern in SC in the same testicular samples. Immunochemical studies clearly showed FSHR expression in fetal and neonatal SC, as well as in adult SC. These findings, which concord with the FSHR transcript expression we also observed in human fetal testis, are also in line with the presence of FSH binding sites in fetal primate testis [START_REF] Huhtaniemi | Follicle-stimulating hormone receptors appear earlier in the primate fetal testis than in the ovary[END_REF]. It is generally accepted that FSH stimulates SC inhibin B (IB) secretion [START_REF] Young | Testicular anti-mullerian hormone secretion is stimulated by recombinant human FSH in patients with congenital hypogonadotropic hypogonadism[END_REF][START_REF] Plant | The functional significance of FSH in spermatogenesis and the control of its secretion in male primates[END_REF][START_REF] Young | Effects of human recombinant luteinizing hormone and follicle-stimulating hormone in patients with acquired hypogonadotropic hypogonadism: study of Sertoli and Leydig cell secretions and interactions[END_REF]. Indeed, the presence of functional FSHR in human neonatal testis is suggested by two recent reports showing an increase in circulating IB in response to the neonatal rise in FSH [START_REF] Bergada | Time course of the serum gonadotropin surge, inhibins, and anti-Mullerian hormone in normal newborn males during the first month of life[END_REF][START_REF] Bougneres | Effects of an early postnatal treatment of hypogonadotropic hypogonadism with a continuous subcutaneous infusion of recombinant follicle-stimulating hormone and luteinizing hormone[END_REF]. Collectively, these results rule out the possibility that the absence of spermatogenesis in fetal and neonatal life is due to a lack of FSHR. AMH, which is responsible for the repression of Mullerian ducts in male fetuses, is measurable in human male umbilical cord serum, as well as in the neonatal period and during infancy [START_REF] Josso | Antimullerian hormone in early human development[END_REF][START_REF] Lee | Measurements of serum mullerian inhibiting substance in the evaluation of children with nonpalpable gonads[END_REF]. The T elevation during puberty correlates with a fall in circulating AMH [START_REF] Rey | Anti-mullerian hormone and testosterone serum levels are inversely during normal and precocious pubertal development[END_REF]. This negative correlation is also observed in boys with central or gonadotropin-independent early puberty, suggesting that T is the main player in AMH down-regulation. Our combined T and AMH assays in umbilical cord, neonatal infants and adult blood provide additional in vivo support for the lack of functional AR expression in SC during the perinatal period. Indeed, the elevated T levels in fetal and neonatal males, in keeping with previous studies [START_REF] Bergada | Time course of the serum gonadotropin surge, inhibins, and anti-Mullerian hormone in normal newborn males during the first month of life[END_REF], are consistent with an increase in TT. However, AMH levels remained elevated during these two developmental stages, in keeping with the absence of AR in SC, as demonstrated here and in another very recent publication [START_REF] Chemes | Physiologic Androgen Insensitivity of the Fetal, Neonatal, and Early Infantile Testis Is Explained by the Ontogeny of the Androgen Receptor Expression in Sertoli Cells[END_REF]. Furthermore, circulating AMH levels were markedly increased in neonates compared to cord blood, owing to the reported FSH surge between these two periods [START_REF] Bergada | Time course of the serum gonadotropin surge, inhibins, and anti-Mullerian hormone in normal newborn males during the first month of life[END_REF][START_REF] Debieve | Gonadotropins, prolactin, inhibin A, inhibin B, and activin A in human fetal serum from midpregnancy and term pregnancy[END_REF]. This AMH increase is probably related to FSHinduced SC proliferation (which increases the AMH-producing cell population), and also to activation of AMH gene transcription through a pathway mediated by cyclic AMP [START_REF] Lukas-Croisier | Follicle-stimulating hormone increases testicular Anti-Mullerian hormone (AMH) production through sertoli cell proliferation and a nonclassical cyclic adenosine 5'monophosphate-mediated activation of the AMH Gene[END_REF][START_REF] Al-Attar | Hormonal and cellular regulation of Sertoli cell anti-Mullerian hormone production in the postnatal mouse[END_REF]. The negative correlation between testicular T and AMH levels is not found in neonatal rodents [START_REF] Al-Attar | Hormonal and cellular regulation of Sertoli cell anti-Mullerian hormone production in the postnatal mouse[END_REF], human fetuses [START_REF] Rey | Anti-mullerian hormone and testosterone serum levels are inversely during normal and precocious pubertal development[END_REF], or normal or cryptorchid human male newborns [START_REF] Bergada | Time course of the serum gonadotropin surge, inhibins, and anti-Mullerian hormone in normal newborn males during the first month of life[END_REF][START_REF] Lee | Measurements of serum mullerian inhibiting substance in the evaluation of children with nonpalpable gonads[END_REF], indicating that testicular T is unable to repress AMH in the fetal and neonatal periods. In newborn rodents [START_REF] Al-Attar | Hormonal and cellular regulation of Sertoli cell anti-Mullerian hormone production in the postnatal mouse[END_REF][START_REF] Majdic | Immunolocalisation of androgen receptor to interstitial cells in fetal rat testes and to mesenchymal and epithelial cells of associated ducts[END_REF] and in adult SCARKO mice [START_REF] Chang | Infertility with defective spermatogenesis and hypotestosteronemia in male mice lacking the androgen receptor in Sertoli cells[END_REF] A: AR was expressed in SMAT1 cells transfected with pcDNA3-hAR plasmid. Proteins (0.5 mg) were immunoprecipitated (IP) with an anti-AR antibody, followed by Western blotting (WB) with the same antibody. AR protein was only expressed in SMAT1 cells transiently transfected with pcDNA3-hAR plasmid. B, C: Relative AMH expression was analyzed by using real-time PCR in control SMAT1 cells (B) and in SMAT1 cells transiently transfected with pcDNA3-hAR plasmid (C). Cells were treated with 10 -5 M forskolin alone or combined with 10 -7 M dihydrotestosterone (DHT) for 24 h. Results are means ± SEM of at least six independent determinations and represent the relative expression compared with basal levels in untreated cells (arbitrarily set at 1). ***, P < 0.001 non parametric Mann-Whitney test. NS: not significant

Figure 4 :

 4 Figure 4: Relationship between AMH and testosterone levels in normal and androgendeficient human males: A: Left Panel: Time course of individual serum AMH levels (pmol/L) in two adult MAIS patients and mean (±SD) serum AMH in 10 adult CHH patients during combined gonadotropins stimulation (recombinant human FSH (rhFSH) and human chorionic gonadodotropin (hCG)). Right panel: Serum AMH levels (pmol/L) were measured in adult, neonatal (20 and 30 post-natal days) and umbilical cord arterial bloods. Results are means ± SEM of at least 19 values. ***, P <0.001 vs adult values, Mann-Whitney test.. B: High AMH immunoexpression in testis of an untreated adult patient with congenital hypogonadic hypogonadotropism (CHH) and two adults with mild androgen insensitivity syndrome (AIS).

Figure 5 :

 5 Figure 5: Androgens repress endogenous AMH expression in SMAT1 Sertoli cells in the presence of AR:A: AR was expressed in SMAT1 cells transfected with pcDNA3-hAR plasmid. Proteins (0.5 mg) were immunoprecipitated (IP) with an anti-AR antibody, followed by Western blotting (WB) with the same antibody. AR protein was only expressed in SMAT1 cells transiently transfected with pcDNA3-hAR plasmid. B, C: Relative AMH expression was analyzed by using real-time PCR in control SMAT1 cells (B) and in SMAT1 cells transiently transfected with pcDNA3-hAR plasmid (C). Cells were treated with 10 -5 M forskolin alone or combined with 10 -7 M dihydrotestosterone (DHT) for 24 h. Results are means ± SEM of at least six independent determinations and represent the relative expression compared with basal levels in untreated cells (arbitrarily

  The receptor is expressed on the Sertoli cell membrane (arrows). Germ cells are negative. D: Representative FSHR mRNA expression in human adult and fetal testis at 28 and 35 weeks. RNA was extracted and reverse transcribed as described in Material and Methods. (See Supplemental Table1for primers). A: Adult. RT-(omission of the reverse transcriptase) and H 2 O are negative controls.

	demonstrating strong AMH expression in SC lacking AR, and the marked fall observed in post-pubertal SC (which express AR) support this assumption. These results also argue against a mechanism in which a change in directional testicular AMH secretion (39) causes the fall in serum AMH after puberty. Patients with androgen insensitivity syndrome or CHH, a pathological condition associated with low testicular T, have abnormally elevated serum AMH levels. As we have previously shown (4, 5) the hCG-driven increase in intratesticular T in the 10 CHH patients studied here, was associated with a marked reduction in the serum AMH level. In contrast, AMH levels were not reduced in the two adult MAIS patients, who received the same combined gonadotropin treatment; on the contrary, they were markedly enhanced, in keeping with an absence of androgen action owing to the lack of AR signaling in SC. Thus, this AMH response pattern in MAIS is similar to that recently reported in two CHH neonates (35) receiving a similar treatment, indicating that in both cases the FIGURE LEGENDS: Figure 1: Immunoexpression of 3β β β βHSD, AR and AMH in human testis: Representative illustrations of immunoreactivity in fetal, neonatal and adult testis. A, B, C: Immunoexpression of 3βHSD in fetal (A), neonatal (B) and adult (C) testis: Enzyme expression is localized to the cytoplasm of Leydig cells (L) in fetal, newborn and adult testis. Sertoli cells and germ cells are negative. D, E, F: Immunoexpression of AR in fetal (D), newborn (E) and adult (F) testis. In fetal (D) and newborn testis (E), nuclear AR expression is restricted to Leydig cells (L) and myoid peritubular cells (arrows). Both Sertoli cells and germ cells are negative. In adult testis (F), AR expression is present in the nucleus of Leydig cells (L), myoid peritubular cells (arrows) and also Sertoli cells (arrowheads). G, H, I: Immunoexpression of AMH in fetal (G), newborn (H) and adult (I) testis AMH is strongly expressed in Sertoli cell cytoplasm in fetal (G) and newborn testis (H). AMH is undetectable in adult human testis. ST: seminiferous tubule Original magnification: x20. failure to repress AMH is related to the absence of functional AR expression in SC. In accordance to these in vivo findings, we obtained the first direct evidence that AMH repression by androgens only occurs when AR is expressed in Sertoli SMAT1 cells. The molecular mechanisms underlying the repressive effect of AR on Figure 2: AMH and AR messenger levels in fetal human testis: A, B: Relative expression of AR (A) and AMH (B) mRNA in 10 fetal human testis obtained at 16 (n=2), 20 (n=1), 21 (n=1), 22 (n=1), 24 (n=2), 25 (n=2) and 38 (n=1) weeks of gestation. (See Supplemental Table 1 for primers). Results were normalized to 18S ribosomal RNA. Relative expression in a given sample was calculated as attomoles per femtomol of 18S RNA. Results are means ± SEM of at least two RT samples run in duplicate, and represent the relative expression compared with control adult testes (arbitrarily 1).	AMH expression remain to be elucidated. AR might directly repress AMH transcription even though no androgen response elements are present in the regulatory regions of the AMH gene. Alternatively, AR might compromise FSHR-mediated AMH stimulation by altering AP2 and NFκB signaling (15). We are currently investigating the functional interaction between AR and FSHR activation cascade on AMH expression using a Sertoli cell model. In conclusion, the absence of androgen receptor expression in Sertoli cells of fetal and neonatal human testis contributes to the lack of germ cell maturation and of anti-Mullerian hormone repression despite strong testicular testosterone biosynthesis.
	Figure 3: FSHR expression in human testis A-C: FSHR protein expression in human fetal (A), newborn (B) and adult testis (C).	, the absence of AR expression in SC is also associated with a lack of AMH repression, suggesting human male newborns. immunohistochemical findings Our that a similar mechanism might exist in

Table 1 :

 1 Summary of AR and AMH immunoexpression in human testisImmunoexpression was classified as: 0: absent or not detectable; Moderate: + and Strong: ++ as described in[START_REF] Mckinnell | Comparison of androgen receptor and oestrogen receptor beta immunoexpression in the testes of the common marmoset (Callithrix jacchus) from birth to adulthood: low androgen receptor immunoexpression in Sertoli cells during the neonatal increase in testosterone concentrations[END_REF].

		Foetal N=46			Newborn n =10			Adult n =10
	Sertoli Leydig Peritubular Sertoli Leydig Peritubular Sertoli Leydig Peritubular AR 0 ++ ++ 0 ++ ++ + ++ ++
	AMH ++	0	0	++	0	0	0	0	0

Acknowledgments

The authors are indebted to Dr Anne-Lise Delezoïde (APHP, Hôpital Robert Debré, Paris, France), Dr Sophie Ferlicot (APHP, Hôpital Bicêtre, Paris, France), Dr Francis Joubert (APHP, Hôpital Necker, Paris, France) and Dr Martine Sinico (CHIC, Creteil, France) for their permission to use testis samples. We thank Annick Ganieux (IFR 93 Bicêtre) for help with the plasmid preparation. This work was supported by funds from Inserm and University Paris-Sud 11 (BQR). KB was the recipient of a fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche, France and from la Fondation pour la Recherche Médicale (FRM).


Sheng Zheng
email: shengzheng@zju.edu.cn

Xue Wu

Eric Lichtfouse
email: eric.lichtfouse@gmail.com

Jing Wang

High-resolution mapping of premature mortality induced by atmospheric particulate matter in China

Keywords: Premature mortality, PM 2.5, Age-and sex-specific population data, Spatial distribution

Air pollution, measured by the concentration of airborne particulate matter with diameter below 2.5 μm (PM 2.5 ), is known to cause globally adverse health effects leading to premature mortality, yet high-resolution spatial distribution of premature deaths is lacking due to the absence of detailed data for the calculation of premature mortality. Here, we mapped the impact of PM 2.5 on premature mortality at high resolution in 2018 using the global exposure mortality model, then we carried out statistical analysis in provincial-level administrative divisions. Results show that the PM 2.5 threshold of 35 μg/m 3 is exceeded in the Xinjiang Uygur Autonomous Region, and in mideastern, highly urbanized and industrial regions of Tianjin, Hebei, Shandong, Henan, Jiangsu, Hubei, Anhui, and Shanghai. Premature deaths due to PM 2.5 exposure in China amounted to 1.44 million, including 510,847 from ischemic heart disease, 471,487 from cerebrovascular disease, 220,837 from chronic obstructive pulmonary disease, 131,321 from lung cancer, and 103,658 from lower respiratory infection. Premature mortality was higher in men, of 879,521 persons accounting for 61%, than women, of 558,629 persons. In provincial-level admin-istrative divisions, high premature deaths were mainly concentrated in the North China Plain, with the highest per capita premature mortality in the Xinjiang Uygur Autonomous Region, Henan, Hebei, and Shandong. Our finer resolution data, together with the age-and sex-specific population data, have improved the estimation of health impacts of PM 2.5 exposure.

Introduction

Coal power plants are common in Asian countries such as China and India [START_REF] Prasad | Influence of coal based thermal power plants on aerosol optical properties in the Indo-Gangetic basin[END_REF][START_REF] Mep | Ambient air quality standards[END_REF]. The emissions from these power plants are one of causes of poor air quality and atmospheric pollution, which have adverse impacts on health, radiative forcing, and climate change [START_REF] Gao | Attribution of aerosol direct radiative forcing in China and India to emitting sectors[END_REF]). In the last decade, airborne particulate matter with diameter below 2.5 μm (PM 2.5 ) has consistently been considered as the major air pollutant in China [START_REF] Cheng | Identifying the spatial effects and driving factors of urban PM 2.5 pollution in China[END_REF]. PM 2.5 pollution has been closely linked to premature mortality due to ischemic heart disease, chronic obstructive pulmonary disease, lung cancer, cerebrovascular disease, and lower respiratory infection [START_REF] Burnett | An integrated risk function for estimating the global burden of disease attributable to ambient fine particulate matter exposure[END_REF]2018). About 6.67 million people died prematurely due to air pol-lution globally in 2019, of which about 1.4 million deaths were caused by PM 2.5 pollution in China [START_REF] Gbd | Global burden of 87 risk factors in 204 countries and territories, 1990-2019: a systematic analysis for the global burden of disease study 2019[END_REF]Risk Factors Collaborators 2020).

The premature mortality caused by PM 2.5 pollution is typically assessed based on different exposureresponse functions such as linear and log-linear models [START_REF] Cohen | Comparative quantification of health risks: global and regional burden of disease attributable to selected major risk factors[END_REF], integrated exposure-response (IER, [START_REF] Burnett | An integrated risk function for estimating the global burden of disease attributable to ambient fine particulate matter exposure[END_REF][START_REF] Li | Specific differences and responses to reductions for premature mortality attributable to ambient PM 2.5 in China[END_REF], and the global exposure mortality model (GEMM, [START_REF] Burnett | Global estimates of mortality associated with long-term exposure to outdoor fine particulate matter[END_REF][START_REF] Tarín-Carrasco | Contribution of fine particulate matter to present and future premature mortality over Europe: a non-linear response[END_REF]. According to the integrated exposure-response model, the PM 2.5 exposure-induced premature deaths in China increased from 1.26 million in 2010 to 2.19 million in 2013, decreased to 1.94 million in 2014, 1.65 million in 2015, 0.96 million in 2016, then slightly increased to 1.05 million in 2017 [START_REF] Xie | Long-term trend and spatial pattern of PM 2.5 induced premature mortality in China[END_REF][START_REF] Li | Estimation of PM 2.5 mortality burden in China with new exposure estimation and local concentration-response function[END_REF][START_REF] Maji | PM 2.5 -related health and economic loss assessment for 338 Chinese cities[END_REF][START_REF] Zou | Air pollution intervention and life-saving effect in China[END_REF]. Later studies showed that the GEMM model, proposed by [START_REF] Burnett | Global estimates of mortality associated with long-term exposure to outdoor fine particulate matter[END_REF], gives better estimates because this model considers a large range of PM 2.5 concentrations, up to 84 μg/m 3 , and is thus particularly adapted for exposure-response investigations in high PM 2.5 pollution areas [START_REF] Bayat | Health impact and related cost of ambient air pollution in Tehran[END_REF][START_REF] Lelieveld | Loss of life expectancy from air pollution compared to other risk factors: a worldwide perspective[END_REF]. Indeed, an epidemiological study of PM 2.5 in China showed better estimates with the GEMM model rather than the integrated exposure-response model [START_REF] Xue | Change in the number of PM 2.5 -attributed deaths in China from 2000 to 2010: comparison between estimations from census-based epidemiology and pre-established exposure-response functions[END_REF].

The estimation of PM 2.5 -induced premature mortality depends on exposure-response functions and data sources such as PM 2.5 , population, and baseline mortality rate. In particular, estimates may vary widely depending on the spatial resolution of the data, notably in urban and suburban areas where population is not evenly distributed. A previous study in China reported PM 2.5 with a rather coarse spatial resolution of 10 km, and population data lacked the use of raster data and were not gender-specified [START_REF] Li | Estimating mortality burden attributable to short-term PM 2.5 exposure: a national observational study in China[END_REF]. Previous investigations of health effects in China are mainly based on statistical studies of whole regions, and lack high spatial resolution of the data [START_REF] Wang | A county-level estimate of PM 2.5 related chronic mortality risk in China based on multi-model exposure data[END_REF][START_REF] Chowdhury | Changing risk factors that contribute to premature mortality from ambient air pollution between 2000 and 2015[END_REF][START_REF] Zheng | Spatial distribution of PM25-related premature mortality in China[END_REF]. It has been shown that the coarse-level mortality statistics would bias the results while the finer resolution of the data opens up the possibility to improve the estimation of PM 2.5 -induced health impacts [START_REF] Goto | Estimation of excess mortality due to long-term exposure to PM 2.5 in Japan using a high-resolution model for present and future scenarios[END_REF][START_REF] Liu | Revisiting the estimations of PM 2.5 -attributable mortality with advancements in PM 2.5 mapping and mortality statistics[END_REF]. Therefore, we studied the spatial distribution of the premature mortality attributable to PM 2.5 pollution using the GEMM model with highresolution gridded PM 2.5 , population data, and provincial baseline mortality rates.

Experimental

Data sources

Gridded population in 2018 is from WorldPop (www. world pop. org), with a spatial resolution of 30 arc seconds: 0.00833°, nearly 1 km at the equator. Demographic data from WorldPop were generated by combining high-resolution satellite remote sensing imagery and census data, and grouped by sex (male, female) and age, including 0-1 and by 5-year up to 80 + . The mapping strategy was sourced from [START_REF] Pezzulo | Sub-national mapping of population pyramids and dependency ratios in Africa and Asia[END_REF].

Concentrations of surface airborne particulate matter with diameter below 2.5 μm (PM 2.5 ) concentrations in China were from the Atmospheric Composition Analysis Group with a spatial resolution of 0.01° × 0.01° (https:// sites. wustl. edu/ acag/ datas ets/ surfa ce-pm2-5/# V4. CH. 03; van [START_REF] Van Donkelaar | Regional estimates of chemical composition of fine particulate matter using a combined geoscience-statistical method with information from satellites, models, and monitors[END_REF][START_REF] Hammer | Global estimates and long-term trends of fine particulate matter concentrations (1998-2018)[END_REF]. The dataset contains ground-based observations of total PM 2.5 concentrations from a network of observatories in China in 2018 and provides combined geophysical-statistical estimates of PM 2.5 concentrations for the national region [START_REF] Hammer | Global estimates and long-term trends of fine particulate matter concentrations (1998-2018)[END_REF][START_REF] Van Donkelaar | Regional estimates of chemical composition of fine particulate matter using a combined geoscience-statistical method with information from satellites, models, and monitors[END_REF].

The baseline mortality rates (BMR) due to five diseases associated with PM 2.5 exposure are from [START_REF] Zhou | Cause-specific mortality for 240 causes in China during 1990-2013: a systematic subnational analysis for the global burden of disease study 2013[END_REF], a dataset that included the number of premature deaths for leading causes in provincial-level administrative divisions (PLADs) in China. The baseline mortality rates for the Taiwan Province were obtained from the Global Burden of Disease (GBD)'s age-standardized data (http:// ghdx. healt hdata. org/ gbd-resul ts-tool).

Global exposure mortality model

The global exposure mortality model (GEMM) was applied to estimate age-related hazard ratios for the five diseases in adults (> 25 years) and lower respiratory infection (LRI) in children under 5 years of age. GEMM revealed the exposure-mortality association and used a large range of concentration intervals (2.4-84.0 µg/m 3 ) [START_REF] Burnett | Global estimates of mortality associated with long-term exposure to outdoor fine particulate matter[END_REF]. The hazard ratio (HR) has been calculated using:

where HR is the hazard ratio of premature mortality attributable to PM 2.5 pollution; β is the exposure-response model coefficient; z is max (0, PM 2.5 -2.4 µg/m 3 ), 2.4 µg/m 3 represents the theoretical lowest exposure concentration; and the set of parameters (α, μ, ν) controls the curve form of the mortality-PM 2.5 relationship [START_REF] Burnett | Global estimates of mortality associated with long-term exposure to outdoor fine particulate matter[END_REF].

Estimation of premature mortality

The formula for calculating the PM 2.5 -attributable premature mortality is:

where ΔMort is the PM 2.5 exposure-induced premature mortality; B is the baseline mortality rates of five specific diseases related to PM 2.5 , including ischemic heart disease (IHD), chronic obstructive pulmonary disease (COPD), lung cancer (LC), cerebrovascular disease (CEV), and lower respiratory infection (LRI); P refers to the exposed population; and HR is the estimated hazard ratios of the five diseases (1)

HR = exp { log (z∕ + 1)∕(1 + exp {-(z -)∕ })} (2) ΔMort = B × P ×
HR -1 HR using Eq. 1. Baseline mortality rates at the provincial level in 2013 are from [START_REF] Zhou | Cause-specific mortality for 240 causes in China during 1990-2013: a systematic subnational analysis for the global burden of disease study 2013[END_REF]. We established estimates according to the hypothesis of unchanged baseline mortality between 2013 and 2018.

Guidelines on PM 2.5 exposure and risk

The WHO air quality guidelines have set four decreasing thresholds of annual average PM 2.5 concentrations 35 μg/m 3 for interim target 1, 25 μg/m 3 for interim target 2, 15 μg/m 3 for interim target 3, and 10 μg/m 3 for interim target 4 (WHO, 2021). Those thresholds are defined as the lowest exposure level of an air pollutant above which adverse health effects would increase. Interim targets refer to incremental steps in reducing air pollution for a specific decrease in health risk.

The Chinese guidelines, named ambient air quality standards (AAQS) (MEP, 2012) set the grade I threshold at 15 μg/m 3 and the grade II threshold at 35 μg/m 3 .

Results and discussion

Spatial distribution of airborne particulate matter

Figure 1 shows the spatial distribution of airborne particulate matter with diameter below 2.5 μm (PM 2.5 ) concentration in 2018 at the provincial level in China. Areas that exceed the standard of 35 μg/m 3 are mainly concentrated in the Xinjiang Uygur Autonomous Region, named 'Xinjiang' for short in the text below, and mideastern areas of Tianjin, Hebei, Shandong, Henan, Jiangsu, Hubei, Anhui, and Shanghai. The main sources of PM 2.5 pollution in the Tianjin-Hebei region, the Yangtze River Delta, and the Shandong Peninsula are traffic emissions, industrial emissions, and construction dust [START_REF] Shan | The pattern and mechanism of air pollution in developed coastal areas of China: from the perspective of urban agglomeration[END_REF][START_REF] Chen | PM 2.5 over North China based on MODIS AOD and effect of meteorological elements during 2003-2015[END_REF]).

The highest PM 2.5 concentration were found in Henan, of 51.3 μg/m 3 , and Tianjin, of 48.3 μg/m 3 . Beijing, Hunan, and Shanxi also had high PM 2.5 concentrations. The less polluted region is the Tibet Autonomous Region with 5.3 μg/m 3 , which is below the fourth WHO target threshold of 10 μg/ m 3 . Other less polluted provincial-level administrative divisions include Taiwan, of 11.6 μg/m 3 , Qinghai, of 12.3 μg/ m 3 , Heilongjiang, of 14.2 μg/m 3 , Hainan, of 14.2 μg/m 3 , and Inner Mongolia Autonomous Region, of 15.0 μg/m 3 . These regions are mainly located at borders or in sparsely populated areas. These findings are in agreement with previous reports [START_REF] Wang | A county-level estimate of PM 2.5 related chronic mortality risk in China based on multi-model exposure data[END_REF][START_REF] Huang | Spatial-temporal distribution characteristics of PM25 in China in 2016[END_REF]. Overall, we found the highest PM 2.5 pollution in the Xinjiang and mideastern areas, and the lowest in less-populated Tibet Autonomous Region.

Premature mortality induced by airborne particulate matter

At the country level, our calculations indicate that the mean total premature mortality caused by PM 2.5 pollution in China in 2018 was 1.44 million, ranging from 0.96 to 1.83 million with 95% confidence. The PM 2.5 exposure-induced premature deaths were:

• 510,847 (465,327) for ischemic heart diseases • 471,487 (233,441-662,819) for cerebrovascular diseases • 220,837 (114,070-309,452) for chronic obstructive pulmonary diseases • 131,321 (84,158-171,076) for lung cancers • 103,658 (60,588-133,134) for lower respiratory infections Ischemic heart and cerebrovascular diseases thus accounted for 68.3% of PM 2.5 pollution deaths.

The distribution of the causes of premature mortality is given in Fig. 2a for the 34 Chinese provincial-level administrative divisions. Ischemic heart diseases and lung cancers were the highest causes in the Shandong province reaching PM 2.5 : airborne particulate matter with diameter below 2.5 μm 51,194 and 13,596 deaths, respectively. Henan showed the highest cerebrovascular diseases premature mortality of 50,918. Premature mortality from chronic obstructive pulmonary diseases and lower respiratory infections peaked at 23,991 in Sichuan and 8858 in Guangdong, respectively. This is because among the 34 provincial-level administrative divisions:

• Guangdong, Shandong, and Henan provinces had the top three highest population In terms of gender, premature mortality for all diseases was higher in men (879,521) than women (558,629), especially for lung cancers, where the premature mortality in men was about 2.8 times that in women (Fig. 2b). The PM 2.5 exposure-induced premature deaths in terms of gender were:

• 303,461 in men and 207,386 in women for ischemic heart diseases • 281,953 in men and 189,534 in women for cerebrovascular diseases • 136,924 in men and 83,913 in women for chronic obstructive pulmonary diseases • 96,576 in men and 34,745 in women for lung cancers • 60,606 in men and 43,052 in women for lower respiratory infections

We compared our results of premature mortality caused by PM 2.5 pollution, of 1.44 million, with previous reports (Table S1). Previous studies found premature mortality caused by PM 2.5 pollution ranging from 0.91 million to 1.83 million. Our result of 1.44 million is very close to the data of 1.43 million by [START_REF] Maji | Substantial changes in PM25 pollution and corresponding premature deaths across China during 2015-2019: a model prospective[END_REF], which is based on the citylevel populations and PM 2.5 concentrations. Our result is also very close to the data of 1.42 million by [START_REF] Giani | Exploring sources of uncertainty in premature mortality estimates from fine particulate matter: the case of China[END_REF], despite the fact that these authors did not take the deaths from lower respiratory infection into account. Their data had a 8 km resolution for PM 2.5 concentration, which is a coarser resolution than our resolution, of 0.01° (nearly 1 km). The lowest estimates of 0.91 million by [START_REF] Maji | Substantial changes in PM25 pollution and corresponding premature deaths across China during 2015-2019: a model prospective[END_REF] are explained because the integrated exposure-response calculations involve a much lower risk ratio [START_REF] Burnett | Global estimates of mortality associated with long-term exposure to outdoor fine particulate matter[END_REF]). The highest estimates of 1.83 million by [START_REF] Ma | Health impact attributable to improvement of PM 2.5 pollution from 2014-2018 and its potential benefits by 2030 in[END_REF] are mainly attributed to different data sources for population and baseline mortality rates. We used the same spatial resolution of 1 km for PM 2.5 concentration as [START_REF] Ma | Health impact attributable to improvement of PM 2.5 pollution from 2014-2018 and its potential benefits by 2030 in[END_REF]. Nonetheless, they used all-age population density data, whereas we used different age ranges. Overall, if we take into account the differences in calculations and data, and the elevated confidence intervals, our results are in agreement with previous work, and show a rather elevated number of premature deaths caused by PM 2.5 pollution.

Spatial distribution of premature mortality

The map of grid-level premature deaths at 1 km resolution shows that the highest mortalities are located in eastern areas, mainly in the urbanized agglomerations downstream of the Yellow River, the eastern part of the Sichuan-Chongqing region, middle reach of the Yangtze River, the eastern coastal region, the central part of Shaanxi and Shanxi, and the western coastal region of Taiwan (Fig. 3a). In terms of specific raster values, the vast majority of grids had less than 5 cases per grid, whereas there were 332 grids (2.41 × 10 -5 of all grids) with more than 50 cases, which mainly located in Tianjin, Shaanxi, Liaoning, Guangdong, Henan, Xinjiang (> 20 grids each). Our findings are roughly in accordance with previous studies [START_REF] Xie | Long-term trend and spatial pattern of PM 2.5 induced premature mortality in China[END_REF][START_REF] Wang | A county-level estimate of PM 2.5 related chronic mortality risk in China based on multi-model exposure data[END_REF]), but the data in this study had finer spatial resolution and were sorted by age groups and gender. The finer spatial resolution could take tiny spatial differences in exposure or mortalities into account and reduce bias in the estimation [START_REF] Vodonos | Estimation of excess mortality due to long-term exposure to PM 2.5 in continental United States using a high-spatiotemporal resolution model[END_REF].

The grid-level map provides a high-resolution distribution of premature deaths based on detailed parameters (Table S1). However, provincial-level administrative divisions are the first-level administrative divisions in China, and air pollution intervention policies in China are based on provincial scales; we have conducted statistical analysis at the provincial scale. The map of premature deaths in provincial-level administrative divisions shows that high premature mortalities are located mainly in the North China Plain, and amount to 132,360 in Henan,128,461 in Shandong,103,337 in Hebei,76,479 in Jiangsu,and 69,874 in Anhui (Fig. 3b). The premature deaths are also high (red legend in Fig. 3b) in Guangdong (88,904) and Sichuan (83,115). The cases in these provincial-level administrative divisions represent 47% of Chinese cases.

The number of premature deaths roughly increases with population and PM 2.5 concentrations (Fig. 3c). The top three provincial-level administrative divisions, Henan, Shandong, and Hebei, with the largest number of deaths had annual average PM 2.5 concentrations over 35 μg/m 3 , with Henan and Shandong both exceeding 45 μg/m 3 . This spatial characteristic is also reflected in existing air pollution control policies. In October 2021, Ministry of Ecology and Environment of the People's Republic of China issued 2021-2022 Autumn and Winter Action Plan for Air Pollution Management, involving Henan, Shandong, Hebei and surrounding areas.

The large population also accounted for the high premature mortality. For example, yearly PM 2.5 concentration was relatively low in Guangdong (23.8 μg/m 3 ), but was considerable premature mortality (88,904) due to its high population. In comparison, Macau, Tibet Autonomous Region, Hainan, Hong Kong, Qinghai, Ningxia Hui Autonomous Region, and Taiwan, which were mainly concentrated in Western China and Southern China, had fairly low case numbers, below 70,000. In order to quantify the relationship among premature deaths, population, and PM 2.5 concentrations, we constructed the multiple regression model for the number of premature deaths using population and PM 2.5 concentrations in 34 provincial-level administrative divisions. The divisions. The red circle indicates the regions with high total premature deaths were concentrated in highly polluted and high population regions. d Spatial distribution of per capita premature mortality. PM 2.5 : airborne particulate matter with diameter below 2.5 μm value of the coefficient of determination (R 2 ) is 0.892 (p value < 0.001), which means that 89.2% of the number of premature deaths can be attributed to population and PM 2.5 concentrations. Specifically, population has a more significant effect on the number of deaths than PM 2.5 , with standardized coefficients of 0.824 (p value < 0.001) and 0.221 (p value < 0.01), respectively.

Since population has an impact on the calculation of premature deaths, in order to eliminate the influence of population, the per capita premature mortality in provincial-level administrative divisions was estimated. The per capita premature mortality was a more direct indicator of PM 2.5 pollution than the total number of deaths, because the correlation between PM 2.5 concentration and per capita premature mortality was greater than that between PM 2.5 concentration and the number of deaths, with the spearman's rank correlation coefficient of 0.64 (p value < 0.0001), and 0.51 (p value < 0.01), respectively. The areas with the highest per capita premature mortality included Xinjiang, Henan,Hebei,and Shandong (14.3,13.7,13.6,and 12.8 per 10,000 persons, respectively) (Fig. 3d), which was consistent with the distribution of provincial-level administrative divisions with the highest PM 2.5 concentrations.

Tianjin, Anhui, and Liaoning also had high per capita premature mortality, although they were relatively lower than the above-mentioned areas. In some provincial-level administrative divisions, the per capita premature mortality was relatively low, despite severe PM 2.5 pollution. For example, Shanghai and Jiangsu had a per capita premature mortality of 6 and 9.2 per 10,000 persons, respectively, but they both had PM 2.5 concentrations above 35 μg/m 3 (Fig. 4). The phenomenon could be attributed to the relatively low baseline mortality rates of these provincial-level administrative divisions. Specifically, ischemic heart and cerebrovascular diseases accounted for 68.3% of all premature deaths. The baseline mortality rates for the two diseases were relatively low in Shanghai and Jiangsu. In men, for example, the baseline mortality rate for ischemic heart disease was the third and seventh lowest in Shanghai and Jiangsu, respectively, and the baseline mortality rate for cerebrovascular disease was the fourth and tenth lowest in Shanghai and Jiangsu, respectively [START_REF] Zhou | Cause-specific mortality for 240 causes in China during 1990-2013: a systematic subnational analysis for the global burden of disease study 2013[END_REF].

Conclusion

We spatially quantified the effects of PM 2.5 on human health based on gridded population and PM 2.5 data. Compared to previous provincial or municipal population data, the data in this study not only had more refined age segmentation but also were sorted by gender. In addition, since the BMR values were based on provincial-level administrative divisions, our overall results were more detailed at the pixel level. Our results show that areas of China with high PM 2.5 concentrations were mainly distributed in the Xinjiang and mideastern regions. With respect to specific causes, ischemic heart disease and cerebrovascular disease were responsible for the majority of PM 2.5 exposure-induced premature mortality. The number of total premature mortalities was about 1.44 million and mortality in all five specific causes was higher in men than women. Moreover, in provincial-level administrative divisions, high premature deaths were mainly concentrated in the North China Plain. Per capita premature mortality was a more direct indicator of PM 2.5 pollution than the total number of deaths. The areas with the highest per capita premature mortality included Xinjiang, Henan, Hebei, and Shandong. These findings require the government to implement more stringent and efficient air quality improvement measures.

Fig. 1

 1 Fig. 1 Annual average of pollution by airborne particulate matter with diameter below 2.5 μm (PM 2.5 ) in China in 2018. The highest pollution levels are observed for the Xinjiang Uygur Autonomous Region and mideastern provincial-level administrative divisions

Fig. 2 a

 2 Fig. 2 a Disease-specific premature mortality caused by pollution by airborne particulate matter with diameter below 2.5 μm (PM 2.5 ) in the 34 provincial-level administrative divisions in China in 2018. b Gender impact of premature mortality. The blue bars represent men and the orange bars represent women. IHD: ischemic heart disease, COPD: chronic obstructive pulmonary disease, LC: lung cancer, CEV: cerebrovascular disease, LRI: lower respiratory infection. PM 2.5 : airborne particulate matter with diameter below 2.5 μm

Fig. 3

 3 Fig. 3 Spatial distribution of premature mortality in 2018 in China at the grid scale (a) and scale of provincial-level administrative divisions (b). c PM 2.5 exposure-induced premature mortality by population and PM 2.5 concentration in 34 provincial-level administrative

Acknowledgements

This research was funded by the National Natural Science Foundation of China (Grant No. 42007194); the MOE (Ministry of Education in China) Project of Humanities and Social Sciences (Grant No. 17YJCZH264); the open fund by Jiangsu Key Laboratory of Atmospheric Environment Monitoring and Pollution Control (KHK1907); and the China Scholarship Council (Grant No. 202006325015).

Supplementary Information

The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s10311-022-01445-6.

Declarations

Conflict of interest The authors declare that they have no conflict of interest in this work.