Vincent Brault

Emilie Lebarbier

Amélie Rosier
email: amelie.rosier@esme.fr

Virginie Stoppin-Mellet

Classification contrainte de signaux, application à l' étude de la prot éine neuronale tau

Keywords: Données fonctionnelles, Classification automatique, Modèle de mélange, paramètres contraints, algorithme ECM Functional data, Model-based clustering, Constrained parameters, ECM algorithm

Ce travail est motivé par une application en neuroscience, en particulier par l'étude du (dys)fonctionnement d'une protéine appelée Tau. L'objectif est d'établir une classification de profils d'intensité, selon la présence ou pas de la protéine et sa proportion monomère ou dimère. Pour cela, nous proposons ici un modèle de mélange gaussienne en un nombre fixé de groupes dont les paramètres de moyennes sont contraints et partagés par les groupes. L'inférence de ce modèle est faite via l'algorithme classique EM. La méthode proposée sera évaluée via des études de simulations et une application sur des données réelles sera effectuée.

Introduction

Ce travail est motivé par une application en neuroscience que nous détaillerons dans un premier de temps avant de présenter la modélisation proposée pour répondre aux problématiques sous-jacentes.

Contexte biologique. La protéine Tau est une protéine présente dans le cerveau qui participe au contrôle de l'architecture et la stabilité des réseaux de microtubules, qui sont essentiels à la propagation des messages nerveux. Des dysfonctionnements de la protéine Tau sont responsables de pathologies graves, comme la maladie d'Alzheimer. Pour étudier la façon dont la protéine Tau contrôle la dynamique des microtubules, l'équipe d'Isabelle Arnal de l'institut Neurosciences de Grenoble a mis au point une méthode permettant de reconstituer in vitro des réseaux de microtubules dynamiques en présence de la protéine Tau, et de les observer en temps réel par microscopie [START_REF] Elie | Tau co-organizes dynamic microtubule and actin networks[END_REF]). A l'issu de ces expériences sont obtenus des profils d'intensité lumineuses au cours du temps. En théorie, ces profils sont des fonctions en escalier décroissantes, comme l'illustre la figure 1, où chaque saut correspond à l'extinction d'une protéine et donc où le nombre de sauts (de même taille) indique le nombre de protéines présentes. Classification contrainte. Etant donné un ensemble de profils, l'objectif statistique est d'établir une classification de ces profils selon le nombre de sauts et leurs tailles. Pour cela, on propose ici un modèle de mélange de distributions gaussiennes dont la moyenne est une fonction constante par morceaux décroissante et différente selon 4 groupes : elle est soit constante (groupe 1), i.e. sans présence de protéine, soit avec 1 ou 2 sauts de taille contrainte : un saut de taille δ pour le groupe 2, deux sauts de taille δ chacun pour le groupe 3 et un saut de taille 2δ pour le groupe 4. La taille des sauts est partagée par tous les profils/signaux tandis que les instants de sauts sont signal-spécifique. Un algorithme EM est utilisé pour estimer les paramètres du modèle.

Modèle

On considère S signaux de taille n s chacun, notés y 1 , . . . , y S avec y s = (y s 1 , . . . , y s n s ) ′ . On suppose que chaque signal y s est une réalisation d'un processus gaussien Y s de taille [n s × 1] dont la moyenne dépend du groupe auquel il appartient. De plus, on suppose l'indépendance inter-signaux et intra-signal. Formellement, on introduit une suite de variables aléatoires indépendantes Z s = (Z s 1 , Z

Y s | Z s k = 1 ∼ N (m s k , σ 2,s k I n s ) for s ∈ [[1 , S]],
où le vecteur de la moyenne de taille

[n s × 1] est pour t ∈ 1, n s m s k (t) =          µ s 1 if k = 1 µ s 2 + δ 1 t>t s 21 if k = 2 µ s 3 + δ 1 t s 31 <t≤t s 32 + 2δ 1 t>t s 32 if k = 3 µ s 4 + 2 δ 1 t>t s 41 if k = 4 ou de façon équivalente m s k = µ s k 1 n s + T s k δ, (1)
où T s k est la matrice d'incidence des instants de sauts, appelés instants de ruptures, pour le signal y s dans le groupe k de taille [n s × 1] :

T s 1 = 0 n s , T s 2 = 0 n s 21 1 n s 22 , T s 3 =   0 n s 31 1 n s 32 21 n s 33   , T s 4 = 0 n s 41 21 n s 42 , (2)
On note t s ki la ième rupture, n s ki = t s ki -t s k(i-1) la longueur du ième segment associé t s k(i-1) + 1, t s ki , avec la convention 0 = t s k0 < t s k1 < t s k2 < t s kI k = n s où I k est le nombre de segments dans le groupe k (I 1 = 1, I 2 = 2, I 3 = 3, I 4 = 2). La moyenne pour les 4 groupes sont représentées en Figure 2.

Les paramètres du modèle sont

Ψ = (π, δ) avec π = (π k ) k=1,...,4 , qui implique tous les signaux, et Φ = (Φ s k ) k,s , où Φ s 1 = (µ s 1 , σ 2,s 1 ) et pour k ≥ 2 Φ s k = (µ s k , σ 2,s k , T s k )
, qui est signal-spécifique et de taille I k + 1 par signal y s dans le groupe k. On note θ = (Ψ, Φ).

Inférence

On propose d'estimer les paramètres du modèle par maximum de vraisemblance. On utlise pour cela l'algorithme classique EM [START_REF] Dempster | Maximum likelihood from incomplete data via the EM algorithm[END_REF]), qui consiste à maximiser l'espérance conditionnelle Q(θ, θ ′ ) de la log-vraisemblance des données complète, log

V (Y, Z; θ) avec Z = (Z s ) s , étant donné Y : Q(θ, θ ′ ) = E θ ′ [log V (Y, Z; θ) | Y ], où E θ ′ [.] est l'opérateur espérance utilisant θ ′ comme paramètre et log V (Y, Z; θ) = S s=1 log 4 k=1 (π k f (Y s ; Φ s k , δ)) Z s k = S s=1 4 k=1 Z s k log(π k f (Y s ; Φ s k , δ)),
f (Y s ; Φ s k , δ) = n s σ s k √ 2π exp - 1 2σ 2,s k ∥Y s -m s k ∥ 2 = n s σ s k √ 2π exp - 1 2σ 2,s k ∥Y s -µ s k 1 n s -T s k δ∥ 2 .
L'algorithme EM est un algorithme itératif combinant, à chaque itération, deux étapes : l'étape E et l'étape M décrites respectivement dans les deux sous-sections suivantes.

Etape E.

Elle consiste à calculer Q(θ, θ (h) ) en utilisant le paramètre courant θ (h) :

Q(θ, θ (h) ) = E θ (h) S s=1 4 k=1 Z s k log(π k f (Y s , Φ s k , δ))|Y = S s=1 4 k=1 τ s k (h+1) log(π k f (Y s , Φ s k , δ)), avec τ s k (h+1) = = P θ (h) (Z s k = 1|Y s ) = π (h) k f (Y s , Φ s,(h) k , δ (h) ) 4 l=1 π (h) l f l (Y s , Φ s,(h) l , δ (h) )
, la probabilité a posteriori d'appartenance du signal s au groupe k.

CM-steps.

L'étape M globale consiste à mettre à jour les paramètres en maximisant l'espérance conditionnelle obtenue à l'étape précédente :

θ (h+1) = arg max θ S s=1 4 k=1 τ s k (h+1) log(π k f (Y s , Φ s k , δ)).
On utilise ici l'algorithme ECM qui découpe la maximisation de Q(θ, θ (h) ) selon θ en étapes CM qui se focalisent sur un paramètre, les autres étant fixés. Les propriétés de convergence de ECM ont été étudiées par [START_REF] Meng | Maximum likelihood estimation via the ecm algorithm : a general framework[END_REF]).

Estimation de π. Les proportions π k sont estimés sous la contrainte 4 k=1 π k = 1. On obtient

π (h+1) k = arg max π Q(θ, π, δ (h) , Φ (h) ) = S s=1 τ s k (h+1)

S

for k = 1, . . . , 4.

Estimation de δ. On obtient

δ (h+1) = arg max δ Q(θ, π (h+1) , δ, Φ (h) ) = arg min δ S s=1 4 k=1 τ s k (h+1) σ 2,(h) k ∥Y s -µ s(h) k 1 n s -T s(h) k δ∥ 2 = S s=1 4 k=2 τ s k (h+1) σ 2,(h) k t T s(h) k (Y s -µ s(h) k 1 n s ) S s=1 4 k=2 τ s k (h+1) σ 2,(h) k t T s(h) k T s(h) k
.

Estimation de Φ. Cette étape se réduit à un problème de segmentation contrainte pour laquelle chaque signal y s est segmenté selon chaque groupe, i.e. en accord avec la contrainte de décroissance associée à chaque groupe. Pour chaque s = 1, . . . , S et k = 1, . . . , 4, h+1) , δ (h+1) , Φ)

Φ s(h+1) k = arg max Φ s k Q(θ, π (
i.e.

(µ

s(h+1) k , σ 2,s(h+1) k , T s(h+1) k ) = arg max µ s k ,σ 2,s k ,T s k Q(θ, π (h+1) , δ (h+1) , µ s k , σ 2,s k , T s k ) = arg max µ s k ,σ 2,s k ,T s k - n s 2 log (2πσ 2,s k ) - 1 2σ 2,s k ∥Y s -µ s k 1 n s -T s k δ (h+1) ∥ 2
On différencie le cas du groupe 1 (pas de saut) des autres :

-pour k = 1, (µ

s(h+1) 1 , σ 2,s(h+1) 1 ) = arg max µ s 1 ,σ 2,s 1 - n s 2 log (2πσ 2,s 1 ) - 1 2σ 2,s 1 ∥Y s -µ s 1 1 n s ∥ 2 ,
et on obtient les estimateurs des moindres carrés suivants

µ s 1 (h+1) = Y s = 1 n s n s t=1 Y s t et σ 2,s(h+1) 1 = 1 n s n s t=1 (Y s t -Y s ) 2 .
-pour k ≥ 2, si les positions des ruptures sont connues, les estimateurs de la moyenne de base et de la variance dans chaque groupe sont simplement :

µ s k (T s k ) = 1 n s ∥Y s -T s k δ (h+1) ∥ 2 and σ 2,s k (T s k ) = 1 n s ∥Y s -µ s k (T s k )1 n s -T s k δ (h+1) ∥ 2 .
L'estimation des ruptures est obtenu par

T s(h+1) k = arg max T s k max µ s k max σ 2,s k Q(θ, π (h+1) , δ (h+1) , µ s k , σ 2,s k , T s k ) = arg max T s k Q(θ, π (h+1) , δ (h+1) , µ s k (T s k ), σ 2,s k (T s k ), T s k ) = arg max T s k - n s 2 log (2π σ 2,s k (T s k )) - 1 2 σ 2,s k (T s k ) ∥Y s -µ s k (T s k )1 n s -T s k δ (h+1) ∥ 2 = arg max T s k - n s 2 log (2π σ 2,s k (T s k )) + 1 = arg min T s k ∥Y s -µ s k (T s k )1 n s -T s k δ (h+1) ∥ 2 ,
et les estimateurs finaux de la moyenne et de la variance sont

µ s(h+1) k = µ s k (T s(h+1) k ) et σ 2,s(h+1) k = σ 2,s k (T s(h+1) k
).

Simulations

Pour les simulations1 , nous avons choisi de prendre S = 100 signaux avec pour chaque signal s une longueur de n s = 100, une probabilité d'appartenance à chacune des classes de 1/4 et une variance de 1. Pour les sauts, nous supposons que δ ∈ {-5, -2, -1, -0.5} et les emplacements des sauts sont choisis au hasard entre 10% et 90% de la série et avec une distance de {0, 0.3, 0.6} × n s + 1 entre les deux sauts du cluster 3. Enfin, les moyennes µ s sont choisies de telles sortes que la moyenne des dernières observations valent 2.

Nous estimons les paramètres à l'aide de l'algorithme EM défini en Section 3 et en prenant 1 ou 10 itérations pour la maximisation et en faisant 1 ou 10 initialisations aléatoires (l'estimation avec la meilleure vaisemblance est alors conservée dans ce dernier cas). Pour évaluer la qualité des résultats, nous regardons l'estimation δ par rapport à sa vraie valeur (voir la figure 3), la distance de Hausdorff des positions des ruptures connaissant les bons clusters (voir figure 5) et le pourcentage d'erreurs de classification (voir figure 4). Les résultats s'améliorent avec le nombre d'initialisations testées, comme attendu, mais pas avec le nombre d'itérations de l'étape M.

On observe que quand la détection de ruptures est facile (δ grand), le saut est bien estimé et les ruptures sont bien positionnées alors que lorsque la détection est difficile (δ petit), le saut est sous-estimé et les ruptures sont moins bien positionnées en particulier pour le cluster 3 (voir figures 3 et 5). En terme de classification, le taux d'erreur augmente quand la taille du saut diminue (figure 4). Comme on peut l'observer en figure 6, seul le cluster 3 est correctement prédit. Pour les séries issues des autres clusters, si la détection est très difficile (δ = -1), toutes les séries (ou quasiment) sont classées dans le cluster 3 et si la détection est un petit peu moins difficile, les séries du cluster 4 sont essentiellement classées en clusters 3 et 4, les séries du cluster 2 dans les clusters 2 et 3, et celles du cluster 1 dans les trois premiers clusters. Dans la suite, nous chercherons à limiter ce surapprentissage.

Figure 1 -

 1 Figure 1 -Deux profils d'intensité lumineuse en théorie.

Figure 2 -

 2 Figure 2 -La moyenne des 4 groupes.

Figure 3 -

 3 Figure 3 -Boxplot des δ-δ ⋆ |δ ⋆ | en fonction de la valeur de δ ⋆ (colonnes) et de la longueur du plateau central (ligne). Pour chaque graphique, nous décomposons suivant s'il y a 1 ou 10 initialisations et 1 ou 10 itérations de l'étape M .

Figure 4 -

 4 Figure4-Boxplot des erreurs de classifications en fonction de la valeur de δ ⋆ (colonnes) et de la longueur du plateau central (ligne). Pour chaque graphique, nous décomposons suivant s'il y a 1 ou 10 initialisations et 1 ou 10 itérations de l'étape M .

Figure 5 -

 5 Figure5-Boxplot des distances de Hausdorff des estimations des ruptures connaissant la bonne classe en fonction de la valeur de δ ⋆ (colonnes) et de la longueur du plateau central (ligne). Pour chaque graphique, nous décomposons suivant s'il y a 1 ou 10 initialisations et 1 ou 10 itérations de l'étape M , et la couleur représente les distances pour les clusters 2, 3 et 4.

Toutes les simulations présentées dans cet article ont été réalisées sur les infrastructures de GRICAD (https://gricad.univ-grenoble-alpes.fr), qui sont supportées par la communauté scientifique de Grenoble.


Amandine Degraeve
email: amandine.degraeve@student.uclouvain.be

Thierry Roumeguere

Gilles Tilmans

Marie-Laura Marotta

Eric Huyghe

Georges Fournier

Antoine Faix

Anne-Françoise Spinoit

Karel Decaestecker

François Herve

Florence Boitrelle

Kanerva Lahdensuo

Lorenzo Tosco

Julien Van Damme

The habits of European urologists in the field of cryopreservation before the urological cancers treatment

Introduction: Treatments against urogenital cancers frequently have fertility sideeffects. The strategy to preserve fertility after oncologic treatments is still a matter of debate with a lack of evidence and international guidelines. The aim of this study is to investigate fertility preservation practices before urogenital cancer treatments and to compare national habits.

Material and methods: An online anonymous survey was

Results: Two hundred twenty-eight urologists from six urological societies in five different countries (Belgium, The Netherlands, Luxembourg, France, Finland) filled out the survey. Three quarter (74%; n = 166) usually propose a cryopreservation before orchidectomy.

In case of oligo/azoo-spermia, the technique performed for the sperm extraction during orchidectomy varies among the sample: 70.5% (n = 160) of the responders do not perform a Testicular Sperm Extraction (TESE) nor a Percutaneous Epididymal Sperm Aspiration (PESA).

The cryopreservation for prostate cancer treatments is never proposed in 48.17%

(n = 105) of responders but conversely it is always proposed in 5.05% (n = 11).

The cryopreservation before bladder cancer treatments is not commonly proposed (67.5%, n = 154).

Conclusion:

Our study showed variable country specific tendencies in terms of fertility preservation in the period of treatment of urological cancers. These differences seem to be related to national guidelines recommendations. Standardization of international guidelines is urgently needed in the field of fertility for urological cancer patients.

K E Y W O R D S

bladder cancer, male fertility, prostate cancer, testicular cancer

INTRODUCTION

In the past few decades, therapeutic advances have improved the long-term survival and prognoses of patients with urogenital cancers. [START_REF] Trottmann | Semen quality in men with malignant diseases before and after therapy and the role of cryopreservation[END_REF] Multimodal cancer treatments are frequently aggressive, and sideeffects can include infertility. Several treatments can adversely affect spermatogenesis such as radiotherapy or chemotherapy [START_REF] Trottmann | Semen quality in men with malignant diseases before and after therapy and the role of cryopreservation[END_REF] and some surgical procedures, like retroperitoneal lymphadenectomy, can impair ejaculation. [START_REF] Matos | Fertility in patients treated for testicular cancer[END_REF] It is not always possible to predict the treatment toxicity due to the patient susceptibility, patient baseline fertility situation, drug characteristics-gonadotoxic effects and type of malignancy. [START_REF] Trottmann | Semen quality in men with malignant diseases before and after therapy and the role of cryopreservation[END_REF] Some patients will become definitively azoospermic or, conversely, will recover partially or totally their spermatogenesis after the treatment. [START_REF] Suzuki | Regeneration of spermatogenesis after testicular cancer chemotherapy[END_REF] For these reasons, the Ethics Committee of American Society for Reproductive Medicine and the American Society of Clinical Oncology have recognized the importance to discuss with patients the potential effect of cancer treatments on fertility, to present options for fertility preservation and to address for fertility preservation. [START_REF] Ferrari | Sperm cryopreservation and reproductive outcome in male cancer patients: a systematic review[END_REF][START_REF] Loren | Fertility preservation for patients with cancer: American Society of Clinical Oncology clinical practice guideline update[END_REF] This is of particular interest for young patients who have not had paternity projects yet and in particular considering that testicular cancer (TC)

is the most frequent cancer in young men (incidence between 15-35 years) who have often long-term survival expectation. [START_REF] Ferrari | Variables affecting long-term usage rate of sperm samples cryopreserved for fertility preservation in cancer patients[END_REF][START_REF] Laguna | EAU guidelines on testicular cancer[END_REF] The European Association of Urology (EAU) recommends cryopreservation for TC and suggests to propose semen preservation and fertility assessment, but no recommendation is given for bladder or prostate cancer. [START_REF] Patrikidou | European Association of Urology Guidelines on Testicular Cancer: 2023 Update[END_REF] Currently, the only way to efficiently preserve the reproductive potential in adult or adolescent male patients remains sperm cry-opreservation. Various assisted reproductive techniques are available. The insemination is possible but in vitro fertilization or intracytoplasmic sperm injection can be preferred due to possible reduction in sperm mobility after freeze-thawing. It is a quiet simple and effective way of preserving fertility potential even in oncologic patients with poor semen characteristics.

We performed a European study about daily oncological practice and fertility management during oncologic treatment. The aim was to assess the information rates and the implementation of cryopreservation before urogenital cancer management (testicular, bladder, prostate) by urologists.

MATERIAL AND METHODS

RESULTS

Population

Two because of the chemotherapy toxicity of and 10% (n = 6) thinks that sperm quality will be better after orchidectomy.

A cryopreservation is proposed more often before orchidectomy if the urologist is focused on andrology (p = 0.021) or on oncologic urology (p = 0.029) and if the clinician is younger than 50 years old (p = 0.0167).

A cryopreservation is proposed more often after orchidectomy in Belgium (57.6%; n = 38) or Luxemburg (50%; n = 3) than in France (11.5%; n = 15), Finland (16.7%; n = 1) or in The Netherlands (6.7%; n = 1) (p < 0.001) (Table 2). There was no statistical difference according to the number of orchidectomy/year between countries (Table 2).

France (59%) and The Netherlands (67%) justify the cryopreservation by following the guidelines compared to the other three countries (Belgium 23.9%; Luxembourg 28.6%; Finland 33.3%) (p < 0.001) (Table 2).

The delay to access the fertility preservation does not explain the cryopreservation after orchidectomy for andrologists (0%) but well for 15% (n = 29) of non-andrologists (p = 0.19). The accessibility to the fertility center is the explanation in 23.8% of Belgian urologists, 28.6% in Luxembourg, and 8.5% in France but not in The Netherlands (0%) and Finland (0%) (p = 0.007) (Table 2).

The sperm quality is considered as better quality after the orchidectomy for 7.5% (n = 5) of the Belgian responders, 1.77% (n = 1) in France and 0% in other countries (no significative difference; p = 0.075). For this variable 50% of uro-oncologists were represented but no andrologists. Half of the responders think that the quality of sperm is better before orchidectomy (44.9%; n = 101) without significant difference for andrologists (56.2%; p = 0.16) or uro-oncologists (42.5%; p = 0.6).

Urologists from France and The Netherlands think significantly more that the sperm quality is better before orchidectomy than after in comparison with the three other countries (p = 0.0055).

Postponing the orchidectomy for a week could be harmful on an for patients < 50 years old, 24.1% (n = 32) for patients < 55 years old and 3% (n = 4) for every patient respectively. The second country for cryopreservation proposal is The Netherlands (46.7%, n = 7), then Belgium (43.3%, n = 29) Finland (33.33, n = 2), and Luxembourg (28.6%, n = 2). The proportion of patients that will benefit from cryopreservation before treatment is evaluated as 0% for the majority of the responders (75.4%, n = 172), around 1%-10% of the patients in 22.8% of the responders (n = 52) and 10%-20% of the patients in 1.3% (n = 3).

The evaluation of the cryopreservation's proportion is not influenced by andrological or oncological practice in each country.

Bladder cancer

The cryopreservation before bladder cancer treatments is commonly not proposed (67.5%, n = 154). A quarter propose a preservation for patients < 50 years old (29%, n = 66), 1.3% for patients < 55 years old (n = 3) and 2.2% for every patient (n = 5). The andrologists have significantly more cryopreservation discussions (50% versus 29.6%). The proportion of cryopreservation in this setting represents 1%-10% of patients in 9.8% of the responders (n = 22) and 10%-20% of the patients in 1% (n = 2), respectively. The proportion of cryopreservation is significatively more important for andrologists

DISCUSSION

The TC, although representing only 1% of all cancers, is the most common cancer in men of reproductive age. With a 10-year survival rate of 98% for non-metastatic forms (73% at 5 years), fertility preservation and management is a major issue. [START_REF] Patrikidou | European Association of Urology Guidelines on Testicular Cancer: 2023 Update[END_REF] The issue of sperm cryopreservation in case of testicular neoplasia seems to be well acquired in our sample but the timing of sperm banking and the technique seems less well defined. A quarter of responders perform the preservation after the orchidectomy. The explanation is the orchidectomy's urgency, especially for onco-urologists. There is no precise time limit in the international guidelines for testicular surgery as long as the time to orchidectomy remains reasonable. [START_REF] Patrikidou | European Association of Urology Guidelines on Testicular Cancer: 2023 Update[END_REF] However, there is an important focus on fertility preservation. [START_REF] Patrikidou | European Association of Urology Guidelines on Testicular Cancer: 2023 Update[END_REF][START_REF] Emmanuel | Expedited radical orchidectomy for testicular cancer: compromising fertility outcomes without oncological benefit?[END_REF][START_REF] Ferlin | Management of male factor infertility: position statement from the Italian Society of Andrology and Sexual Medicine (SIAMS): endorsing organization: Italian Society of Embryology, Reproduction, and Research (SIERR)[END_REF] The urologists from France and The Netherlands propose more cryopreservation before orchidectomy. Those results could be related to an easier accessibility to cryopreservation centers in those countries, like the "Centre de conservation des oeufs et du sperme" (CECOS) in France. [START_REF] Walschaerts | Sperm cryopreservation incidence in men with testicular cancer: towards a stabilization in testicular cancer incidence? Results from the CECOS network[END_REF] The justification for sperm banking before the orchidectomy is also more framed by the guidelines follow up in those two countries. [START_REF] Murez | French AFU Cancer Committee Guidelines-Update 2022-2024: testicular germ cell cancer[END_REF][START_REF]Kwaliteitsnormen testiscarcinoom[END_REF] The risk factors of TC are: cryptorchidism, hypospadias, decreased spermatogenesis, and sub-fertility or infertility, familial history of TC among first-degree relatives, and the presence of contralateral tumor. [START_REF] Patrikidou | European Association of Urology Guidelines on Testicular Cancer: 2023 Update[END_REF] TC is 20 times more frequent in infertile men. [START_REF] Walsh | Increased risk of testicular germ cell cancer among infertile men[END_REF] Only 50% of patients coming for sperm cryopreservation in the context of TC have a normal sperm concentration. [START_REF] Rives | The semen quality of 1158 men with testicular cancer at the time of cryopreservation: results of the French National CECOS Network[END_REF] A cryopreservation before orchidectomy is a real opportunity to analyze patient semen quality and identify azoospermia or severe oligo-zoospermia. [START_REF] Ferlin | Management of male factor infertility: position statement from the Italian Society of Andrology and Sexual Medicine (SIAMS): endorsing organization: Italian Society of Embryology, Reproduction, and Research (SIERR)[END_REF][START_REF] Jacobsen | Gonadal function and fertility in patients with bilateral testicular germ cell malignancy[END_REF] It may be beneficial to address patients for fertility preservation earlier in the course of TC treatments to propose per-operative TESE or PESA if a severe spermatogenesis impairment is funded. A pre-operative fertility conservation could also offset the risk of the non-functioning remaining testicle. [START_REF] Patrikidou | European Association of Urology Guidelines on Testicular Cancer: 2023 Update[END_REF][START_REF] Jacobsen | Gonadal function and fertility in patients with bilateral testicular germ cell malignancy[END_REF] About 10% of the specialists think that the sperm quality will improve after orchidectomy however it is debated. The semen concentration can decrease after orchidectomy, considering however the lack of evidence, and the contralateral testicle remaining non-functional. [START_REF] Ostrowski | Infertility with testicular cancer[END_REF] On the other hand, TC specific factors induce worsening of semen Fertility preservation plays an important role for the quality of life. [START_REF] Moody | Fertility managment in testicular cancer: the need to establish a standardized and evidencebased patient-centric pathway[END_REF] Orchidectomy is the best moment to collect sperm via TESE or PESA in case of azoospermia or oligoasthenozoospermia. [START_REF] Blecher | Onco-Testicular Sperm Extraction (oncoTESE): a contemporary concept review and report of Australian sperm retrieval rates and fertility outcomes[END_REF] Unfortunately, 70% urologists seem to be also more familiar with this intraoperative procedure as explicitly stated in their national guidelines and less precisely in the European guidelines. [START_REF] Laguna | EAU guidelines on testicular cancer[END_REF][START_REF] Murez | French AFU Cancer Committee Guidelines-Update 2022-2024: testicular germ cell cancer[END_REF][START_REF]Kwaliteitsnormen testiscarcinoom[END_REF] The very important reason to perform a PESA in combination with the TESE is the epididymal sperm even if only 12.3% of the responders perform the two procedures. The spermatozoa acquire their fertilizing ability and forward mobility properties during epididymal transit. [START_REF] Sullivan | The human epididymis: its function in sperm maturation[END_REF] The interactions between infertility and TC are complex. The testicular dysgenesis syndrome is a pre-existing risk factor for TC, there is a local effect of the tumor and additional adverse effects such as stress and depression. [START_REF] Záková | Sperm cryopreservation before testicular cancer treatment and its subsequent utilization for the treatment of infertility[END_REF] The testicular tumor will have both a local effect on spermatogenesis with greater disturbances in the tissues in contact with the tumor but may also cause a disturbance of spermatogenesis in the contralateral testicles in the case of a large tumor. [START_REF] Matos | Fertility in patients treated for testicular cancer[END_REF] The mechanisms of action involved are the hormonal secretion by the tumor of beta-human chorionic gonadotropin (β-HCG) and alphafetoprotein (α-FP) causing an increase in intra-testicular estradiol or the disturbance of the hypothalamo-hypophyseal axis. [START_REF] Ostrowski | Infertility with testicular cancer[END_REF] The tumor can also induce the development of anti-sperm antibodies. The complementary treatments for TC are chemotherapy (mainly bleomycin, etoposide, vinblastine) and retroperitoneal lymph node dissection (RPLND) or radiotherapy. [START_REF] Patrikidou | European Association of Urology Guidelines on Testicular Cancer: 2023 Update[END_REF] The platinum salts cross the blood-testicular barrier to actively target dividing cells, with a significant effect on spermatogenesis (oligo-azoospermia). [START_REF] Moody | Fertility managment in testicular cancer: the need to establish a standardized and evidencebased patient-centric pathway[END_REF] In the majority of cases, a recovery of spermatogenesis within 2 years is observed and can be complete with recovery of mean semen parameters after a certain time from the end of the treatment. [START_REF] Murez | French AFU Cancer Committee Guidelines-Update 2022-2024: testicular germ cell cancer[END_REF][START_REF] Drechsel | Reproductive ability in survivors of childhood, adolescent, and young adult Hodgkin lymphoma: a review[END_REF] The RPLND can affect the ejaculation and cause retrograde ejaculation or anejaculation even with a nerve sparing approach (15%). [START_REF] Murez | French AFU Cancer Committee Guidelines-Update 2022-2024: testicular germ cell cancer[END_REF] The radiation therapy will primarily target the retroperitoneal lymph nodes. Testicular radiation is the main cause of testicular insufficiency. The effect is dose dependent with permanent azoospermia at 16-18 Gray and Leydig cell failure at > 20 Gray. [START_REF] Patrikidou | European Association of Urology Guidelines on Testicular Cancer: 2023 Update[END_REF] In the absence of pre-orchidectomy conservation, it is essential to offer a sperm banking after orchidectomy and before complementary treatments.

The vast majority of responders does not talk about sperm cryopreservation for prostate or bladder cancer patients older than 55 years old. Practitioners are more likely to discuss fertility before prostate cancer or bladder cancer management if the patient is younger than 55 years old (47% for prostate and 30% for bladder cancer). Delayed parenthood is constantly increasing worldwide due to various socioeconomic factors. [START_REF] Zacchini | Delayed parenthood and its influence on offspring health: what have we learned from the mouse model †[END_REF] The median men's age at the birth of their first child is around 31 years old in Luxemburg and The Netherlands. This age is around 28 years old in Finland, France, and Belgium. Five percent of men have children after the age of 45 years old. [START_REF] Degraeve | The habits of European urologists in the field of cryopreservation before the urological cancers treatment[END_REF] The cryopreservation proportion is low among prostate and bladder cancer patients (1%-10% of the patients). It is certainly due to the age of these cancers' incidence. The demography evolution could justify a preventive approach and an information about fertility preservation for younger patient. Once again, the most sensitive countries to the issue seem to be French, Dutch urologist, or andrologists.

To extrapolate the data results in other countries, we need study less unbalanced towards Belgium and French responders and responders from other European countries.

CONCLUSION

Our study showed variable country specific tendencies in terms of fertility preservation in the period of treatment of urological cancers.

These differences seem to be related to national guidelines and recommendations. International guidelines are urgently needed in the field of fertility for urological cancer patients. It is of utmost importance to study the potential benefit of semen preservation before orchidectomy for future fertility of TC patients.

20472927, 0 ,

 0 Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.13577 by Inrae -Dipso, Wiley Online Library on [18/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License than urologists: 22% versus 7.8% and 3.1% versus 0.5%, respectively (p = 0.015).

  quality and the orchidectomy might lift off these local and general disturbances. Sperm recovery to an improved spermatogenesis needs about 2-3 years. Knowing the peak of incidence of TC in the third decade of life for non-seminoma (NSGCT) and mixed germ cell tumor (GCT), and the fourth decade for pure seminoma (ST), this period of time, without any certainty of recovery, could be detrimental to procreation projects. Given the advances in treatment, more patients with urogenital cancer are surviving and looking to return to the normal life.

  of the responders do not perform this extraction. The sperm extraction is more implemented in France, The Netherlands, by andrologists and by urologists performing more than 10 orchidectomy per year. The specialists who are specialized in TC care and fertility are obviously more concerned about the cryopreservation. Once again, French and Dutch

  Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License vasectomy. There were five questions about cryopreservation during vasectomy reversal. It included 14 questions about urogenital cancers (testicular, prostate, and bladder cancer) and fertility preservation: number of each cancer, proposition of cryopreservation before management, timing of the cryopreservation for TC, reason of the timing (Table1). The section concerning cryopreservation and vasectomy or vaso-vasostomy was discussed in a previous article.[START_REF] Degraeve | Société Belge d'Urologie (SBU), the Belgische Vereniging voor Urologen (BVU), the Association Française d'Urologie (AFU), the Nederlandse Vereniging voor Urologie (NVU), the Société d'Andrologie de Langue Française (SALF), the[END_REF]

	2.1	Statistical analysis
	Statistical analyses were performed using SPSS software version 25
	(SPSS Corp., Somers, New York). The p-value < 0.05 was considered
	statistically significant. Binary variables were expressed as count and
	proportion. Comparison of parameters between countries were made
	using the Pearson's chi-squared test, Fisher's exact test, and binomial
	test when possible. Comparison between urologists with or with-
	out andrological training was made using only Pearson's chi-squared
	test.
		From January to June 2021, we offered to participate in an online
		anonymous survey to different European urological societies from
		12 countries: Ireland, United Kingdom, Sweden, Finland, Denmark,
		The Netherlands, Belgium, France, Spain, Portugal, Italy, Germany. Six
		scientific societies accepted to submit the survey to their members
		by e-mail: The Société Belge d'Urologie (SBU), Belgische Vereniging
		voor Urologen (BVU), the Association Française d'Urologie (AFU), the
		Société d'Andrologie de Langue Française (SALF), The Nederlandse

Vereniging voor Urologie (NVU), and the Finnish Urological Society (FUS).

The 31-items questionnaire included seven demographic questions about urologists: country, region, age, status, gender, sub-specialty, and type of center. There were five questions about cryopreservation and 20472927, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.13577 by Inrae -Dipso, Wiley Online Library on [18/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on

Cryopreservation at the time of urogenital cancers, vasectomy and vasectomy reversal Epidemioly

		Cryopreservation before		Cryopreservation after
		orchidectomy (n)		orchidectomy (n)	p-value	Odds ratio
	<50 years old	102		26	0.0167	/
	>50 years old	33		15
	No andrology practice	137		55	0.021	0.258
	Andrology practice	29	endourology, other 3		(0.08-0.88)
	How old are you? No oncological practice	115	Age	31	0.029	1.96
	What gender? Oncological practice	51	Man, Woman 27		(1.06-3.62)
	In what type of center do you practice urology? Belgium 28	University Center, Public peripheral center, Private peripheral center 38 <0.001 /
	Oncology Luxembourg	3			3
	Approximately how many oncologic orchidectomy do you France 116	Number/year 15
	perform per year? Finland	5			1
	Do you perform cryopreservation for TC? The Netherlands 14	Yes/No	1
	When do you perform cryopreservation for TC? Timing of cryo not according to the	Before orchidectomy; after orchidectomy and before chemotherapy; at the start of chemotherapy; after chemotherapy; other Timing of cryo according
	Why do you perform cryopreservation at this specific guidelines (n)	-Because it is recommended by the guidelines to the guidelines (n) p-Value	Odds ratio
	moment? Belgium	51	-Because I think that orchidectomy is more urgent than the fertility -Byhabits 16 <0.001 /
	Luxembourg	5	-Because of the delay to get access to fertility preservation 2
	France	53	-Because I think that quality of sperm will be better AFTER orchidectomy 76
	Finland	4	-Because I think that quality of sperm is better BEFORE orchidectomy -Because I think that chemotherapy is NOT TOXIC for fertility 2
	The Netherlands	5	-Because I think that chemotherapy is TOXIC for fertility 10
		Delay to access to	-Other Delay to access to
	What is the most urgent for you?	fertility preservation is	Orchidectomy, Fertility preservation fertility preservation is
	Do you think that postponing the orchiectomy for a week not an obstacle (n)	Yes/No an obstacle (n)	p-value	Odds ratio
	to perform cryopreservation could be harmful from an No andrology practice 164		29	0.019	0
	oncological point of view? Andrology practice	32			0
	In case of oligo/azoo-spermia, which technique do you use for sperm extraction during orchidectomy? Belgium 51	-No extraction -Testicular Sperm Extraction (TESE) 16	0.007	/
	Luxembourg	5	-TESE+ Percutaneous Epididymal Sperm Aspiration (PESA) 2
	France	119	-Other 11
	In which testis do you perform sperm extraction? Finland 6	-Both	0
	The Netherlands	15	-In contra-lateral testis only -In the tumor-bearing testicle only 0
	About how many significant prostate cancers do you	Number/year
	diagnose per year?
	Do you discuss cryopreservation with your patient with	PESA. There is a significant difference between urologists without -Always
	significant prostate cancer?		-Rarely, only if it is a patient < 50 years old andrology practice and andrologists who practice much more a TESE -Rarely, only if it is a patient < 55 years old
			extraction during the orchidectomy : 12.8% versus 43.8% (p < 0.0001) -Never
	What proportion of your patients benefit from	(Table 2). No difference was found between the two populations for 0%; 1%-10%; 10%-20%; 20%-30%; other
	cryopreservation before prostate cancer treatment?			TESE with PESA (12.3% for urologists and 12.5% for andrologists)
	About how many muscle invasive bladder cancers do you	(Table 2). The results show that the combination TESE + PESA is much Number/year
	diagnose per year?				more used in The Netherlands (40%, n = 6) and France than in other
	Do you discuss cryopreservation with your patient with	countries. Three countries performed only TESE (Finland 33.3%, Lux--Always
	muscle invasive bladder cancer?		embourg 28.6%, and Belgium 26.9%). Cryopreservation practice varies -Rarely, only if it is a patient < 50 years old
			between countries (p = 0.003). -Rarely, only if it is a patient < 55 years old
			-Never
	What proportion of your patients benefit from
	cryopreservation for bladder cancer before treatment?

oncological point of view for 23.3% of the responders and significantly more for oncologist (36.3%; n = 29, p = 0.0006) but significantly less in France (12.8%; n = 17) than in other European countries (p = 0.0002) In case of oligo/azoo-spermia, the technique performed for the sperm extraction during orchidectomy varies among the sample: 70.5% (n = 160) of the responders do not perform a Testicular Sperm Extraction (TESE) nor a Percutaneous Epididymal Sperm Aspiration (PESA), 17.2% (n = 39) perform only a TESE and 12.3% (n = 28) a TESE and TESE or combination of TESE + PESA are significatively more implemented when the number of orchidectomy/year is > 10: 17.1% versus 60% and 11.1% versus 40%, respectively (p = 0.0073).

When an extraction is done, it can be done in both testicles, in the tumor-bearing testis or in the contralateral one. Among urologists who perform this technique (51%, n = 116), 19% prefer to do it in the TA B L E 1 Online survey questionnaire.

Where do you practice urology? Country

What is your status? Urologist, Andrologist, other What is your specialty? General urology, Onco-urology, Functional urology, Andrology, Pediatric urology, 0%; 1%-10%; 10%-20%; 20%-30%; other Vasectomy/Vaso-vasostomy: The section concerning cryopreservation and vasectomy or vasovasostomy was discussed in a previous article

(9)

. 20472927, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.13577 by Inrae -Dipso, Wiley Online Library on [18/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License TA B L E 2 Study results.

Sperm better before orchidectomy (n) Sperm NOT better before orchidectomy (n) p-Value Odds ratio

	No andrology practice	110	83	0.16	1.7
	Andrology practice	14	18		(0.8-3.6)
	No oncological practice	78	67	0.6	0.86
	Oncological practice	46	34		(0.5-1.5)
	Belgium	44	23		/
	Luxembourg	5	2
	France	62	68
	Finland	5	1
	The Netherlands	8	7
		Postponing orchidectomy	Postponing orchidectomy
		is not harmful (n)	is harmful (n)	p-Value	Odds ratio
	No oncological practice	124	24	0.0008	2.94
	Oncological practice	51	29		(1.56-5.52)
	Belgium	43	24	0.0002	/
					(Continues)

20472927, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.13577 by Inrae -Dipso, Wiley Online Library on [18/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License TA B L E 2 (Continued)

Postponing orchidectomy is not harmful (n) Postponing orchidectomy is harmful (n) p-Value Odds ratio

	Luxembourg	5		2
	France		116		17
	Finland		4		2
	The Netherlands	7		8
						TESE +
			No extraction (n)	T E S E ( n)	PESA (n)	p-Value	Odds ratio
	No andrological practice	146		25	24	<0.0001	/
	Andrological practice	14		14	4
	Belgium		42		18	7	0.003	/
	Luxembourg	5		2	0
	France		103		14	15
	Finland		4		2	0
	The Netherlands	6		3	6
	⩽10 orchidec/year	143		34	22	0.0073	/
	>10 orchidec/year	0		3	2
				Contro-
			No sperm	lateral testis	Both testis	Tumor-bearing
			extraction (n)	extraction (n)	extraction (n)	extraction (n)	p-Value	Odds ratio
	Belgium		22	28	8	9	0.0266	/
	Luxembourg	4	2	0	1
	France		74	31	19	9
	Finland		4	2	0	0
	The Netherlands	8	1	3	3
	tumor bearing testis, 55.1% in the contralateral one, and 25.9% in both
	testicles. There is a significant difference between andrologists who
	perform more bilateral extraction (43.5% vs. 21.5%) or more tumor-
	bearing extraction (21.7% vs. 18.3%) and less contralateral extraction
	only (34.8% vs. 60.2%) (p = 0.0028). There was no statistically signif-
	icant difference considering oncological sub-specialties. There was a
	difference among countries with more contralateral extraction rates
	in Belgium and tumor-bearing extraction rates in The Netherlands
	(p = 0.0266) (Table 2).
	3.1.2	Prostate cancer
	The cryopreservation for prostate cancer treatments is never pro-
	posed in 48.17% (n = 105) of responders but conversely it is always
	proposed in 5.05% (n = 11). It is only proposed for patients < 50

years old in 29.82% (n = 65) or proposed for patients < 55 years old in 16.97% (n = 37). There is no difference between andrologists or oncological specialists but there are different practices among countries (p = 0.0346). The French specialists propose significantly more cryopreservation before prostate cancer treatments: 30.1% (n = 40)

20472927, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.13577 by Inrae -Dipso, Wiley Online Library on [18/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.