José Carlos Frutos Dordelly

Mohamed El Mankibi

Letizia Roccamena

Gabriel Remion

Jesus Arce Landa

Experimental analysis of a PCM integrated solar chimney under laboratory conditions

Keywords: Solar chimney, passive ventilation, energy storage, PCM

The objective of this work is to experimentally investigate the impact of integrating a Phase Change Material (PCM) on the performance of two different laboratory solar chimney prototypes, rendering it a viable option for yearlong use. It has been stated that solar chimneys can provide constant ventilation and increase air quality in a building. This study aims to provide a different approach in means of improving the current performance of solar chimneys as, nowadays, it is mostly pursued through the modification of the inclination, the air gap size or the inlet/outlet dimensions. The solar chimney prototype mainly analysed in this work is built with 2 cm plywood plates with a thermal conductivity of 0.15 W/mK with a volume of 3.50 × 1.00 × 0.30 m. After a 6 hour charge period, a mean ventilation rate above 70 m 3 /h can be achieved with a relative low gain of 550 W/m 2 provided by a series of 7 halogen lamps directed towards an effective collector area of 3.00 m 2 . The results obtained in this work show that PCM integration provides a higher ventilation rate and a slower decrease during ventilation only phases (6 hour discharge), where the halogen lamps do not provide any energy to the solar chimney. Overall, the implementation of paraffinic PCMs in solar chimneys could be an economically viable option for hybrid design solutions to create a healthy indoor environment within residential buildings through renewable solar energy.

Introduction

Passive ventilation relates to natural ventilation systems which exploit natural resources such as wind or thermal buoyancy to set off an air current to and from an indoor space (can be induced between other things through solar energy). The goal of these type of systems is to control the temperature and enhance the refreshment rate of the air of such spaces. In general, a building experiences passive ventilation through the opening and closing of windows, but other alternatives, such as solar chimneys, can achieve this same effect with a higher performance. Solar Chimneys consist of a channel used to evacuate hot air from a building through a closed conduct at a Email addresses: carlos.frutosdordelly@entpe.fr (José Carlos Frutos Dordelly), mohamed.elmankibi@entpe.fr (Mohamed El Mankibi), letizia.roccamena@entpe.fr (Letizia Roccamena), gabriel.remion@entpe.fr (Gabriel Remion), jesuso@cenidet.edu.mx (Jesus Arce Landa) higher elevation. By means of the greenhouse effect, temperature rises across the channel and induces a thermal up-draft. Most current designs [START_REF] Arce | Experimental study for natural ventilation on a solar chimney[END_REF], have made modifications to the basic design to add elements such as a glazing or an opposite collector wall (Figure 1), both of which capitalize on the incoming solar energy to improve the performance of a simple-build solar chimney.

Solar chimneys were conceived for the main purpose of improving the thermal comfort conditions. Nowadays, construction elements such as the solar chimney, must attain to a number of health, safety and comfort regulations [START_REF] Duffie | Solar Engineering of Thermal Processes[END_REF][START_REF]The Emissions Gap Report 2017 -A UN Environment Synthesis Report[END_REF][START_REF]Standard 55 -Thermal environmental conditions for human occupancy[END_REF][START_REF]Standard 62.1 -Ventilation for acceptable indoor air quality[END_REF] and must be affordable both energetically or economically. Additionally, in the interest of reducing global warming from an estimated 4.5 • C (by 2100) to less than 2.7 • C and decreasing greenhouse gas emissions per capita by 9% by 2030 [START_REF]Adoption Of The Paris Agreement. Adoption of the Paris Agreement[END_REF], solar chimneys can represent a viable alternative to mechanical systems or even other better known passive systems such as trombe walls or wind towers. The reliability of solar chimney lies in its capacity to provide a constant ventilation rate in a househould. In order to maximize said ventilation rate of solar chimneys, some authors such as Khanal and Lei [START_REF] Khanal | An experimental investigation of an inclined passive wall solar chimney for natural ventilation[END_REF] analysed the effects of tilt on the mass flow rate of a solar chimney. The experimental results stated that the temperature distribution across the air gap is not dependent on the inclination angle of the chimney, however, the airflow velocity is greatly influenced by this factor. They stated that for a solar chimney with a 0.70 m absorber height and 0.10 m air gap width at an inclination angle of 6 • and input heat flux of 500 W/m 2 can produce sufficient ventilation for a 27 m 3 room based on ASHRAE standards. On the other hand, the optimal inclination angle, according to Sakonidou et al [START_REF] Sakonidou | Modeling of the optimum tilt of a solar chimney for maximum air flow[END_REF], depends on the available irradiation where the maximum air flow varies in a rather narrow range between 65 • and 76 • whereas for maximum irradiation (900 W/m 2 ) it varies between 12 • and 44 • .

In the interest of maximising the effectiveness of solar chimneys, several approaches have been addressed. Chantawong et al [START_REF] Chantawong | Investigation on thermal performance of glazed solar chimney walls[END_REF] modified the conventional architecture of the solar chimney to integrate glazed walls and ensure direct radiation on the collector wall. Nevertheless; this type of technology is exclusively profitable during the day, as long as solar energy increases the temperature of chimney and the air inside it. Thus far, the induced up-draft produced by the solar chimney has been experimentally studied mostly in countries where there is a high and constant solar gain such as Egypt, Iran or Algeria to mention a few [START_REF] Hosien | Effects of the geometrical and operational parameters and alternative outer cover materials on the performance of solar chimney used for natural ventilation[END_REF][START_REF] Saifi | Experimental study and simulation of airflow in solar chimneys[END_REF][START_REF] Shahreza | Experimental and numerical investigation on an innovative solar chimney[END_REF] (see Figure 2). It has been stated that temperature registered in the control volume can cool down faster and heat up slower with the addition of such technology [START_REF] Tan | Influences of ambient air speed and internal heat load on the performance of solar chimney in the tropics[END_REF].

Only certain authors focus on the nocturnal or seasonal potential of this type of technology. Some studies have focused on the coupling of alternative systems to enhance the cooling potential or the ventilation rate [START_REF] Dai | Enhancement of natural ventilation in a solar house with a solar chimney and a solid adsorption cooling cavity[END_REF][START_REF] Liu | Experimental study of the chimney effect in a solar hybrid double wall[END_REF][START_REF] Al Touma | Performance assessment of evaporatively-cooled window driven by solar chimney in hot and humid climates[END_REF], however, the present study focuses on the capitalization of this technology for nocturnal or seasonal ventilation. Martí-Herrero et al [START_REF] Martí-Herrero | Dynamic physical model for a solar chimney[END_REF] analysed a 0.24 m thick concrete collector wall, with high thermal inertia, for nocturnal ventilation and numerically demonstrated the capabilities of a solar chimney when the system has a higher thermal inertia.

According to literature, the most common suggestion towards the prolonged utilization of solar chimneys, is the modification of the collector wall's construction material and/or dimensions. This driving element has been an important subject of interest in different works.

Studies such as the one by Ryan et al [START_REF] Ryan | Experimental study of the influence of collector height on the steady state performance of a passive solar air heater[END_REF] studied the effects of diverse collector sizes on the efficiency of the system (0.50, 1.00 and 2.00 m). They reported that for a lightweight collector wall, the height has a positive effect on the performance of the chimney, independent of the aspect ratio, stating that maximising the channel height and depth is recommended for higher flow rates. Depending on the incident solar energy, the results suggest that the optimal aspect ratio lies between 0.1 and 0.2.

Several works demonstrate the typical weather conditions of countries such as Mexico and Spain [START_REF] Matsumoto | Global solar irradiation in north Mexico city and some comparisons with the south[END_REF][START_REF] Arce | Thermal performance analysis of a solar chimney , based on the experimental study of the main driving variables in a physical prototype[END_REF] have high incident solar radiation which can be employed for this type of renewable technology. The expertise of the thermal inertia and the resulting mass flow rate have been studied in previous works such as the ones by Bansal et al [START_REF] Bansal | Solar chimney for enhanced stack ventilation[END_REF], Khanal et al [START_REF] Khanal | An experimental investigation of an inclined passive wall solar chimney for natural ventilation[END_REF] and Mathur et al [START_REF] Mathur | Experimental investigations on solar chimney for room ventilation[END_REF] to name a few. In the same way, the capitalization of solar chimneys after sunset or during colder seasons is still a barrier to be raised.

Phase Changing Materials (PCMs) have been recently analysed and employed due to their energy storage capabilities. PCMs represent a viable solution for the building sector [START_REF] Bahrar | Numerical and experimental study on the use of microencapsulated phase change materials (PCMs) in textile reinforced concrete panels for energy storage[END_REF][START_REF] Roccamena | Experimental test bed design and development for PCM-water exchangers characterization[END_REF], when compared to other options such as sensitive thermal storage, however the incorporation to building components remains a relatively new field which must be researched further researched under different scenarios. Solar chimneys have been employed as a coupled system of other ventilation systems, however the pursuit for Zero-Energy Buildings requires the continuous development and optimization of systems. PCMs represent an extra investment over the construction price. In order for this type of technology to be viable, it must yield better results than common construction materials and/or simplify its design.

Objective

The energy storage particularity of PCMs can be coupled with the collector wall characteristics in order to prolong the uptime of the solar chimney. Solar chimneys with PCM have been experimentally analysed by Li et al.,and Amori and Mohammed [15,[START_REF] Liu | An experimental study on the thermal performance of a solar chimney without and with PCM[END_REF][START_REF] Amori | Experimental and numerical studies of solar chimney for natural ventilation in Iraq[END_REF] under laboratory conditions where the PCM was stored in a 0.04 m thick steel container with RT42 (melting temperature 42 • C) and an unspecified paraffin wax (melting temperature 56.16 • C), respectively. Both studies were carried out on a 2 × 1 m solar chimney with an air gap (distance between the glazing and the collector wall) of 0.20 and 0.15 m respectively.

The design of the solar chimney prototype presented in this study is based on the previous work of Arce et al. [START_REF] Arce | Thermal performance analysis of a solar chimney , based on the experimental study of the main driving variables in a physical prototype[END_REF] which represents a greater air volume. The aim of this work is to analyse the addition of the paraffinic RT44 PCM panels (melting temperature 44 • C) in a solar chimney under laboratory conditions for a polystyrene and a plywood-based structure. The interest of the study relies in the analysis of such an available and customer-accessible technology.

Furthermore, the results will be analysed in future works against the in situ results obtained.

Experimental Set-up

The solar chimney prototypes were tested under laboratory conditions, subjected to the temperature of the experimental site. Wind speed was neglected from the analysis and a constant approximate energy of 550 W/m 2 was provided using 7 halogen lamps. These halogen lamps were positioned perpendicular to the glazing at a distance of 0.50 m to ensure that the incident energy would cover the totality of the collector wall (3.00 m 2 ). No energy variations were considered during charging phases. In order to ensure the simulation of different experimental scenarios, a controlled inlet volume was employed. This volume is presented in section 3.3. For both prototypes, the system was located in a closed environment, without direct solar radiation. The prototype V1.0, henceforth addressed as SCV1, served the purpose of analysing the behaviour of PCM panels on a simple-build solar chimney. This first laboratory prototype was built with 5cm-wide polystyrene plates, supported by an aluminium beam structure (0.03 m cross section). The inlet of the chimney was fixed at 0.5 × 0.075m, while the circular outlet has a diameter of 0.16m. The dimensions of the prototype are noted in Table 1. The first solar chimney prototype allowed an initial characterization of the system and the advantages of PCM panels on a structure with no thermal inertia (solely driven by the PCMs) however, the construction materials of this prototype were not representative of a real building component. The second version of the laboratory solar chimney prototype was developed after the analysis of the first laboratory and the in-situ re- sults [START_REF] Dordelly | Active Solar Chimney ( ASC ) -Numerical and experimental study of energy storage and evaporative cooling[END_REF]. The development of the second prototype was based on two main reasons:

Solar Chimney Prototype V1.0

Solar Chimney Prototype V2.0

1. The representation and analysis of an actual building component integrated with PCM panels and, 2. mainly, the future comparative study against the insitu solar chimney results.

The results obtained during the first laboratory experimental campaign and the first cycles with the second prototype, allowed the integration of some best-practices for the optimisation of the experimentation cycles. In order to propitiate the phase change and take better advantage of the incoming irradiance, the panels were painted matte black. This modification was carried out in relationship to the in-situ experimental campaign, where the panels received the same treatment and after the analysis of the surface temperature results of the SCV1 discussed in section 6.1. Indeed, the results obtained with the first prototype evidenced the incomplete fusion of the PCM, thus hampering the performance of the system. In addition to this modification, an additional layer of insulation was added behind the plywood layer where the panels were placed. This additional layer, was added to avoid panel heat loss to the exterior and to ensure the panel temperature rise above the fusion range of 44 • C.

The second prototype of the solar chimney, hereinafter called SCV2, was developed according to the design of the PSA (Solar Platform of Almeria) in-situ solar chimney in Spain [START_REF] Arce | Experimental study for natural ventilation on a solar chimney[END_REF]. Due to laboratory size restrictions, the SCV2 was shortened by 0.50 m in relation to the the solar chimney at the PSA. In terms of PCM panel placement, this reduction represents one less row of panels in comparison to the PSA solar chimney experimentation. Figure 4 displays the SCV2 at the ENTPE laboratory. The second prototype is built with 2 cm plywood boards. The size of the inlet is 0.8 × 0.08 m while the outlet has the same size as the SCV1 (0.0201m 2 ). A 0.007 m thick pane of glass covers the front of the chimney and works as a door to the inside of the chimney.

The volume behind the solar chimney is a cubic volume of 4.68 m 3 . This volume, just as the one of the SCV1, serves the purpose of defining a control volume at the inlet of the chimney where an inlet temperature can be defined. This structure is connected to the controlled inlet volume (section 3.3) is connected through a 1.190 × 0.94 m opening (see Figure 4). The volume is built with the same type of plywood as the solar chimney.

Controlled Inlet Volume

A cooling unit was coupled to the solar chimney prototypes (Figure 3) in order to simulate (during certain experimental cycles) a controlled temperature in an inner volume. This device can maintain a controlled atmosphere at a low temperature (lower than ambient temperature), up to a minimum temperature of 0 • C, and was directly connected to the prototype control volume's left side (see Figures 3 and4). Although the external temperature is an uncontrollable variable, the possibility of creating a ∆T allows different experimental scenarios. The cooling unit is located inside an insulated volume which is connected to the chimney's rear volume. In turn, this volume is linked to the chimney via the inlet located in the lowest part of the system. The cooling unit represents a volume of 3.18 m 3 which coupled with the chimney's rear body constitutes a total volume of 7.40 m 3 for the polystyrene prototype and 7.58 m 3 for the plywood prototype.

PCM Selection

Several options were taken into consideration for thermal energy storage, however, the Rubitherm RT44 panel rose amongst other options (Figure 5) due to four main factors: the availability, fast delivery, encapsulation provided by the supplier and the relative low cost per panel. Indeed, this work prioritized a viable solution for the everyday consumer and not only for a scientific application. The working temperature range of the PCM must maximize the time the chimney is at a high temperature to ensure ventilation. The temperature range of 40 to 44 • C was selected based on the available experimental and environmental data of the stand alone solar chimney in Almeria [START_REF] Arce | Experimental study for natural ventilation on a solar chimney[END_REF] from which this study is based on. Finally, from an experimental stand point, the PCM layer must take into consideration safety regulations, and must be a light durable container without making a complex or heavy-weight prototype. The instrumentation on the first prototype was limited due to parallel ongoing experimentations with the GHB, however, 15 surface temperature sensors, 2 wind speed sensors, 2 air temperature sensors and 2 heat flux plate sensors were available for the experimentation. Platinum thermoresistance (PT100, 1/10 DIN) sensors are used to record surface temperature of the panels via a four-wire connection. These sensors consist of a very small sensing element embedded in a slim rubber substrate. Likewise, air temperature measurements were carried out with PT100 sensors, however, the rubber protection is replaced with a stainless steel sheath. The PT100 sensors have an uncertainty of 0.1 • C. Delta Ohm hot wire transmitters (HD403TS series) are employed for the continuous measurement of the inlet and outlet air speed. This type of sensors have a 4 -20 mA output with is converted to 0.0 -5.0m/s. The speed measurements are precise up to ±0.2m/s and requires a 24V power supply.

Finally, HFP03 heat flux plate sensors from the company Hukseflux were selected to analyse the heat flux provided by the halogen lamps. This type of sensor estimates the heat flux of the surface it is mounted on, in W/m 2 . This type of sensor has a 6% uncertainty linked to the calculation of the heat flux when dividing the plate's output voltage by the sensitivity of the thermopile (passive sensor). This heat flux plate does not require a power supply.

The acquisition system is composed of four different National Instruments acquisition modules. The information relative to the acquisition modules is presented in Table 4. The surface temperature acquisition modules have an approximate uncertainty of ±0.2 • C.

The SCV1 can accommodate 9 panels in a horizontal position in the surface perpendicular to the heat source, and 6 panels in a vertical arrangement on each of the lateral walls of the chimney as seen in Figure 8. The twelve panels found on the lateral panels served the purpose of analysing the effects of PCM panels when placed parallel to the heat source and their capability to The surface temperature sensors were distributed in such way as to analyse the temperature distribution on the three walls of the solar chimney, at different levels. Along the height of the chimney, the sensors were allocated evenly according the availability of these at three different levels (Figure 6). The first prototype allowed the analysis of PCM panels both perpendicular and parallel to the heat source.

SCV2 Instrumentation

The equipment employed for the analysis of the SCV2 was mostly the same as the one used during the experimental campaign of the first prototype. Three air temperature PT100 sensors, both Hukseflux HFP03 heat flux plate sensors as well as both Delta Ohm hot wire transmitters to measure wind speed were used for the SCV2. The two main changes were the surface temperature sensors and the acquisition modules. SFSC50 flexible temperature probe with resistive element from C2AI were employed to register surface temperatures across the chimney. These class A PT100 sensors have an uncertainty of ±0.15 • C + 0.002|T |. All sensors (air temperature, surface temperature, heat flux and air speed) relay information to a wireless acquisition module from Delta Ohm. These HD35ED wireless data logging systems allow the continuous monitoring of different physical quantities such as temperature, humidity, air speed, heat flux, CO 2 levels, between others. The information is then sent to the nearby computer in order to be stored and displayed.

Contrary to the first solar chimney prototype, the SCV2 did not allocate PCM panels in the lateral walls, thus the sensors were distributed on the surface perpendicular to the heat source at three different levels and over the three rows of panels, as shown in Figure 11. The row distribution of the sensors was chosen to ensure the surface temperature throughout the level was consistent.

Experimental Protocol

In order to define the performance of a solar chimney with and without PCM panels, an experimental protocol was established. This protocol serves three main purposes:

1. To evidence the differences between the current solar chimney and the PCM integrated solar chimney, under laboratory conditions.

2. To demonstrate the capabilities of phase changing materials, to extend the use of a solar chimney in the absence of a heat source.

3. The validation of a future numerical model.

Figure 12 shows the protocol used for the experimentation. The experimental protocol is composed of seven consecutive phases of 6 hours each (0.25 days), completing a full cycle in 1.75 days. Phases depend on the state of the outlet (open/closed) thus allowing the flow of air for natural ventilation, and heating provided by the halogen lamps (on/off), which simulate solar radiation. The lamps are evenly distributed over the 3.00 m 2 glass surface of the chimney and provide a constant approximate maximum heat flux of 550 W/m 2 during the charging phases.

In a more detailed explanation, phase 1 corresponds to the initialization of the system. In this stage, the outlet is closed and there is no heat source present. Phase 2 was designed as a way to charge the PCM panels in order to guarantee the melting process of the PCMs. Ventilation is neglected in this phase to assure the temperature raise. Phase 3 allows air circulation by opening the outlet and maintaining the heat source. This phase represents the normal utilization of a solar chimney. Phase 4 removes the heat source while allowing air circulation. This phase was designed to exemplify the behaviour of a solar chimney when solar radiation is hampered or for after sun-down hours. Phase 5 reinitializes the system and prepares it for the last two phases. Phases 6 and 7 work in the same way as phases 2 and 4; however, both conditions (opening and heat source) are not activated simultaneously.

Results & Discussion

Outlet mass flow rate, air gap temperature and temperature difference between inlet and outlet, are some of the most important quantities for the design of a solar chimney. These variables allow the definition of the thermal and aerodynamic performance of the solar chimney. In order to take advantage of the full potential of the PCM integrated solar chimney, the collector wall's surface temperature needs to surpass the temperature range of 40 -44 • C, where PCMs exhibit the phase transformation. When this temperature range is not surpassed, the capabilities of PCMs are not met. This section presents the results obtained throughout the different experimental cycles under different operating conditions. The results presented in this section adhere to the nomenclature presented in Figure 13. Results corresponding to the the SCV1, do not mention the prototype number, on the other hand; the SCV2 mention include the prefix P2. The second part of the nomenclature specifies the experimental cycle. The third part specifies the addition of PCM. The number found at the end corresponds to the completed sequence of the experimental protocol for the same cycle. Finally, for every type of result, an extra section is added to specify the results presented (e.g. MFR -Mass Flow Rate). The results shown below were carried out under two different conditions: input temperature set by the controlled inlet volume and no input temperature, thus following the exterior temperature. The difference between both setups is discussed in this section. The results presented in these sections correspond to the cycles presented in Table 5.

Surface Temperature Results

The effects of PCM panels on the surface temperature distribution is particularly noticeable in the results of the first prototype. In terms of surface temperature variations, there are two main modifying factors between the two prototypes: the material, and the insulation. Indeed the first prototype had only a single layer of polystyrene with limited thermal inertia while the second prototype was made of laminated plywood planks with an additional insulating polystyrene layer to avoid heat loss. The lack of thermal inertia of the first system evidences the potential of PCMs.

Regarding the SCV1, the surface temperature results when not integrated with PCM, show a temperature distribution that reaches a difference of up to 25 • C, where the temperature increase is directly dependant on the halogen lamp input. Results show that for a polystyrene-based prototype, temperature increases with height. For a set input temperature (Figure 14), the surface temperature rises by approximately 25 • C from the initial temperature, during charging only phases. When the prototype is subjected to the external temperature (Figure 16), this difference can go up to 35

• C.
The experimental conditions shown in Figure 14 were achieved when the prototype was placed in the first laboratory were the external temperature was controlled by the HVAC system. The mean external temperature registered during the experimental campaign C5NoPCM was of 19.5 • C with variations not exceeding 2.0 • C. On the other hand, the surface temperature results corresponding to C7NoPCM follow the behaviour of the external conditions which and show a dependence on these (Figure 16). The surface temperature difference between the bottom and top sensors differs from cycle to cycle and the operating conditions but it is generally 10 ± 2 • C throughout the experimental campaigns.

When PCM panels are added to the SCV1, this temperature difference is reduced and the overall surface temperature across the chimney revolves around the same level, only varying by approximately 5±2 • C (collector wall surface). The thermal inertia provided by the PCMs to the system is represented in the dynamic of the surface temperature evolution of Figure 15a. Contrary to the NoPCM cycles, the surface temperature is maintained from Phase 3 of the experimental protocol (ventilation + heat source) to Phase 4 (ventilation only) in most PCM cycles. In general, temperature drops only occur once ventilation is neglected and the heat source is turned off. The system is capable of retaining part of the energy provided by the halogen lamps however a complete fusion is not guaranteed for every panel. Due to the overall heat loss and the lack of proper insulation, the bottom panels in most of the cycles of the SCV1 reach the lower end of the melting range 40 • C but do not surpass 44 • C. Nevertheless, the addition of PCM panels hampers the wall heat loss and enhances the system's performance regardless of the incomplete fusion of some of the PCMs.

The results obtained with the SCV1 showed the effects of heat loss on this system. PCM experimental cycles retain the energy provided by the halogen lamps during the ventilation only and initialization phases better than their counterparts. Indeed from t = 0.5 to t = 0.75, the solar chimney without PCMs experiences a temperature due to the opening of the exhaust, while PCM cycles maintain a quasi-stable temperature. From t = 0.75 to t = 1.00, where the protocol goes through the ventilation only phase, PCMs lose 50% of the stored capacity after approximately 3 hours and reach initial state after the initialization phase starts. When a higher external temperature influences the cycle (Figure 17), the PCMs maintain a higher temperature. From the charging only phase at t = 0.25 to the next charging phase at t = 1.25 (24 hours) several PCM panels stay above the 40 • C lower limit.

Moreover, the surface temperature results of the second laboratory prototype were more consistent due to the modifications and best-practices mentioned in section 3.2. Unlike the results obtained with the first prototype, the SCV2 experiences less heat loss throughout the cycle, thus less abrupt temperature drops than the ones obtained with the SCV1 for NoPCM cycles. The influence of the experimental protocol can still be clearly appreciated in the surface temperature evolution of the figures presented for the SCV2 and the temperature difference between peak and dip can reach 30 • C when the inlet temperature is set to 18 • C, and the external conditions reach 37.5 • C.

Unlike the SCV1 results, the second prototype exhibits a less dispersed temperature distribution. Some cases, as the one shown in Figure 19a register a temperature decrease once the heat source is turned off, followed by minor temperature increase due to the rise of the external temperature. Even though, the system experiences less heat loss, the protocol is still well defined in the dynamic of the experimental cycle. During NoPCM cycles, after the activation phases (for 0.50 ≤ t ≤ 0.75 and 1.00 ≤ t ≤ 1.25), the surface temperature drops approximately to the inlet temperature level.

On the other hand, PCM cycles in the SCV2 evidenced the influence of inlet temperature during the experimental cycle. It can be observed (Figures 2122) that as soon as the heat source is taken away, the lower panels' temperature drop, mainly due to them not reaching melting temperatures. Panels located at higher altitudes, which have reached complete fusion, release the stored energy during the 6 hours of ventilation only phases. Figure 23 shows the behaviour of the system when solely influenced by the external temperature. The overall surface temperature of the chimney revolves around the melting/congealing temperature range and this represents a higher thermal potential.

Finally, the PCM results of the SCV2 evidence two main points: firstly, the incomplete fusion of the PCM severely hampers the potential of phase changing materials in the overall performance of the chimney. Naturally, when the panels do not reach the fusion range, the system behaves as the NoPCM cycles, with a slight higher thermal inertia. In general, temperatures do not attain the same temperature peaks as the NoPCM cycles since some of the energy is employed for the phase transformation, however; they follow the experimental protocol in a similar fashion. Secondly, the lower PCM panels, even though they reach the fusion temperature range, fail to surpass 44 • C which translates into an incomplete energy storage. These panels exhibit lower temperature drops similar to the results obtained without PCM. The rest of the system surpasses this temperature range and maintains a higher mean temperature throughout the test.

Overall, when comparing SCV1 cycles subjected to similar operating conditions, NoPCM cycles reach temperature peaks 20 % higher than the PCM counterparts. During ventilation only phases, however; PCM cycles outperform their NoPCM cylces by maintaining a temperature 33 % higher during the 6 hours of the protocol phase. On the other hand, the SCV2 temperature peaks for NoPCM and PCM cycles reach approximately the same magnitude but the mean temperature throughout the cycle is significantly higher.

Air Temperature Results

The experimental protocol was ran under two different conditions. As explained in section 3.3, the controlled inlet volume can set an operating temperature at the volume connected to the chimney and thus, at the inlet level. Through the establishment of an inlet temperature, a ∆T between inlet and outlet can be propitiated. In general, a greater ∆T will lead to a higher mass flow rate. The first prototype evidenced the effects and potential of PCMs over a system with low thermal inertia. This section focuses on the results obtained with the second prototype (SCV2) due to the better construction material and thus, the consistency of the results.

The six cycles presented in this section are compared according to the operating and external conditions. Cycles P2C1NoPCM-2 and P2C1PCM-2 were carried out without the additional insulation layer and with a fixed inlet temperature. Cycles P2C3NoPCM-2 and P2C2PCM-2 were carried out with the additional insulation layer and with a fixed inlet temperature. Finally, cycles P2C4NoPCM-3 and P2C5PCM-2 were carried out with the additional insulation layer and without a set inlet temperature but following the external temperature. Overall, air temperatures tend to follow the behaviour and magnitude of surface temperatures.

The variation between the setpoint temperature of cooling unit and the inlet temperature depends on the external temperature, however for the cycles discussed in this section it is generally 5 ± 1.5 • C. Once the charging phase starts, this temperature rises but never above 7.5 • C. When the cooling unit is not employed, inlet temperature follows external temperature behaviour and the experimental protocol has little effect on its magnitude.

Cycles where the chimney was not insulated and an inlet temperature was set (Figure 24) present similar dynamics. Inlet temperature is severely influenced by the setpoint temperature of the cooling unit and its thus maintained at a quasi stable temperature of 23 ± 3 • C, experiencing increases during charging only phases. Air temperature measured at the other two heights (1.60 and 3.20 m) have close behaviours with a temperature difference across the cycle of 5 • C at most. The results presented in Figure 21 in section 6.1 show the top level panels reaching the lower limit of the melting range, thus engaging energy storage, which results in higher air temperatures at outlet level showed in Figure 24b. Indeed, outlet air temperature at 1.00 ≤ t ≤ 1.25 follows a less pronounced drop than the air temperature at a height of 1.60 m.

It is clear that the incomplete fusion of PCM panels makes the system behave in a similar fashion (Figures 24b and25a), however; when PCM completes the phase change, air temperatures present less variations and lesser drops. Cycles without insulation, or without PCM panels, register temperature decreases of up to 20 • C, while PCM cycles reduce this difference to approximately 12.5 • C in the most critical conditions. PCM cycles, maintain a higher air temperature throughout the cycle compared to the NoPCM cycles.

Results evidence the influence of inlet temperature on the air temperature. Indeed, the utilization of the cooling unit hampers the system's thermal performance and does not allow the PCM panels below 2.50 m to reach melting range. Nevertheless, the energy provided by the system allows the system to reach temperatures of 40 ± 5 • C, where this temperature is maintained above 35 • C in PCM cycles for 24 hours.

The temperature difference achieved with the solar chimney as well as the consistent results throughout the experimental campaigns, suggests that the system has a great potential to use the heated air inside the chimney to be recirculated in order to reduce heating loads in a building. No air quality study was carried out during the experimental campaigns, however, the air inside of the chimney can be classified as class 2 according to the ASHRAE standard 62.1 [START_REF]Standard 62.1 -Ventilation for acceptable indoor air quality[END_REF], where the air contains a moderate amount of contaminants, a mild sensoryirritation intensity or mildly offensive odours. This type of air is not necessarily harmful but since the chimney does not receive constant maintenance, dust and other particles could be found in the air. Nevertheless, the temperature potential could be coupled with other systems.

Mass Flow Rate Results

The results shown in Figures 27 and28 demonstrate the influence of the different stages of the experimental protocol on the outlet mass rate. For the following figures, the section highlighted in grey represents phases 4 and 7 of the experimental protocol. These phases show the impact of the PCMs since they correspond to ventilation only phases. Phase 4 comes after the charging phase [START_REF] Amori | Experimental and numerical studies of solar chimney for natural ventilation in Iraq[END_REF] where energy is supplied to the system preventing air flow, and the activation phase (3) where both the heat source and the ventilation are present. On the other hand, phase 7 comes after a charging only phase not going through an activation phase [START_REF] Arce | Thermal performance analysis of a solar chimney , based on the experimental study of the main driving variables in a physical prototype[END_REF]. The results shown in the following figures show the NoPCM cycles in black and the PCM cycles in blue.

The SCV1 without PCM panels shows dramatic mass flow rate drops as soon as the protocol enter phases 4 and 7. The polystyrene structure provides no thermal inertia to the system, which causes a drop in outlet mass flow rate as soon as the heat source is withdrawn. The mass flow rate during phase 3 is very similar for the first set of results when the external temperature is below 25 • C. The maximum mass flowa registered without PCMs was 90 m 3 /h, however this value drops to nearly 0 as soon as the heat source is turned off.

In turn, SCV1 results with PCM panels display a slow decrease of mass flow rates between phases. When panels do not reach the melting range, the system behaves similarly to the NoPCM solar chimney. Naturally, when a greater number of panels surpass the PCM fusion temperature range, the system exhibits a greater mass flow rate (Figure 28). This improvement is mainly due to the higher external temperature between cycles being compared. During phase 3 of the experimental protocol, when there is a heat source present and the outlet allows airflow, the chimney presents the highest values. C1PCM-1 achieves an approximate mean mass flow rate of 87.50 m 3 /h, while C3PCM-2 reaches 187.5 m 3 /h. Phases 4 and 7 which correspond to ventilation only phases, vary according to the percentage of RT44 in liquid state. The results from the latter figure demonstrate the potential of the PCM panels by maintaining a mean mass flow rate of 112.50 m 3 /h. These results are obtained despite the incomplete fusion of several panels and the incapability of the rest to surpass the 44 • C. The results obtained with the SCV1 evidence the potential of PCM panels, however; a more refined analysis was necessary in order to validate this assumption. The SCV2 was developed in order to analyse the true impact of PCM panels on a building component. SCV2 mass flow rate results display a similar behaviour and magnitude during phase 3 (activation) for both NoPCM and PCM values (approximately 82.50 m 3 /h). The differences in the mass flow rate during this phase for NoPCM and PCM cycles depend on the external temperature. Indeed, as temperature rises, the mass flow rate rises as well for cycles subjected to external temperature (Figure 31). The performance of the system during phases 4 and 7 of NoPCM cycles depends greatly on the temperature difference between inlet and outlet, as shown in the aforementioned figure, particularly noticeable in phase 7.

PCM cycles for the SCV2 show a consistent performance of the system regardless of the operating conditions. Results demonstrate that the system is capable of maintaining an approximate mean ventilation rate of 60 m 3 /h during the 6 hours of the experimental protocol phase but the performance is halted as the phase reaches the end. A lower inlet temperature hampers the performance of the system, which translates to a reduction in the outlet mass flow rate (Figure 30). Furthermore, when the system is exposed to the external temperature, the system displays an increased performance. This is mainly due to the higher surface temperatures and the greater number of PCM panels achieving fusion. The major difference between the exit airflow is observed for the SCV1 results. The results demonstrate that the incomplete fusion of the PCM panels make the system perform without any improvements. The main difference is found during the ventilation only phases (4 and 7) when the thermal inertia of the PCMs slightly increases the airflow from 15.59 m 3 /h and 18.92 m 3 /h to 17.25 m 3 /h and 26.64 m 3 /h. Once the temperature increases and allows the fusion of the PCM panels the the PCM integrated SCV1 outperforms its counterpart by 247 % during phase 3 and a mean ventilation rate of 110.04 m 3 /h and 122.39 m 3 /h during phases 4 and 7 where the non PCM version has a near 0 m 3 /h airflow.

Similarly, the results SCV2 display an approximate airflow increase of 365 % during phase 3 when the system is not properly insulated and the chimney suffers a great heat loss. Once the heat loss is limited through the addition of insulation, both NoPCM and PCM cycles reach similar peaks and behaviours during phase 3 and the airflow difference is appreciated during ventilation only phases. Phase 4 seems to behave in a very similar manner however, phase 7 always displays a higher ventilation rate. When setting an inlet temperature, due to the incomplete fusion of all the PCM panels, the system only experiences an increase from 43.03 m 3 /h to 53.51 m 3 /h. On the other hand, when subjected to external conditions, the system exhibits the greatest performance increase going from 60.82 m 3 /h to 34.93 m 3 /h.

Conclusions

The laboratory results of the SCV1 showed the interest of integrating Phase Change Materials to the solar chimney, to improve the average ventilation rate in the presence of a heat source, and to ensure a progressive decrease otherwise. These results were obtained despite the incomplete fusion of the material within the panels, which suggests better performance when the complete phase change is achieved. The PCM panels work as expected, absorbing available energy during each charging phase and releasing once the source is withdrawn. In general, the results with phase change materials show a higher average flow rate in all phases of the experiment. It is important to mention that these results are achieved despite the incomplete fusion of the materials which means an increase in performance at higher temperatures for a simple build solar chimney with a low thermal inertia. The results of the first prototype are an example of the potential of PCMs as separate component.

Overall, PCMs exhibit a greater performance compared to NoPCM cycles, even during tests which do not reach the fusion range. The consistency in performance of PCM cycles suggests that the addition of PCM panels has no negative impact on the system and can improve its efficiency depending on external conditions, mainly higher irradiance and higher external temperatures. The series of temperature and mass flow rate results are obtained with a maximum incoming heat flux of 550 W/m 2 . Incident solar radiation in countries such as Mexico, Spain or the south of France can reach higher peaks than the ones simulated in these experimental campaigns. Higher radiation can lead to higher surface temperatures and higher air temperatures which would aid the whole group panels to achieve a full phase transformation. Overall the results of both the SCV1 and the SCV2 exhibit a greater thermal performance of the PCM integrated solar chimney. Indeed the temperature peaks might not reach the same highs but they maintain a stable high temperature across the experimental cycle.

The laboratory results presented in this section evidence the following: 1. The system's overall performance is highly dependent on the thermal characteristics of the chimney walls. Indeed, heat can reduce not only surface temperatures, but airflow for NoPCM and PCM cycles as well. The influence of the construction materials on the solar chimney's performance cannot be neglected.

2. The addition of PCM panels gives consistency to the system despite the operating conditions. PCM panels provide thermal inertia, and the additional layer avoids heat loss across the chimney.

3. PCMs provide ventilation throughout the 6 hours of ventilation only phases after the panels have been charged. This period is limited due to the nature of the experimental protocol, however, it could be extended according to the external conditions, or through the extension of ventilation phases.

4. The PCM integrated solar chimney is capable of maintaining a surface temperature higher than 40 • C for the 6 hours of the experimental phase (in absence of a heat source) when subjected to external temperature. This represents a 33 % higher temperature over its counterpart during the same period.

5. Inlet air temperature severely influences the PCM performance. PCMs stabilize the air temperature across the experimental cycle at a higher temperature. These results suggest that the inlet location and the connection to the household must be further analysed.

6. During charging phases, both NoPCM and PCM cycles have similar mass flow rate results, however; during ventilation only phases, the PCM integrated solar chimney can reach provide a mean mass flow rate higher than 60 m 3 /h for the whole duration of the experimental phase (6 hours).

7. The RT44 aluminium panels represent an accessible solution for thermal energy storage, improving the performance of the solar chimney in the absence of a heat source.

With the results obtained during these laboratory experimental campaigns, future works will look towards development of a numerical tool capable of reproducing the behaviour of a PCM integrated solar chimney. Furthermore, the addition of PCM on the collector wall will be studied under environmental conditions at the PSA in Spain, as well as and the optimal operating parameters for these energy storage devices.

Figure 1 :

 1 Figure 1: Schematic diagram of a simple solar chimney with vertical inlet subjected to environmental conditions.

Figure 2 :

 2 Figure 2: Location of experimental (red), numerical (blue) and numerical & experimental (purple) solar chimney studies.

Figure 3 :

 3 Figure 3: First solar chimney laboratory prototype developed at ENTPE connected to the cooling unit (glazed volume to the left of the prototype).

Figure 4 :

 4 Figure 4: Second solar chimney laboratory prototype developed at ENTPE

Figure 5 :

 5 Figure 5: RT44 aluminium panel employed for the both solar chimney prototypes.

  latent and sensible heat in a temperature range of 35 • C to 50 • C.

Figure 6 :

 6 Figure 6: Instrumentation employed for the solar chimney SCV1 displaying (blue) surface temperature sensors, (green) air temperature sensors, (orange) Heat flux plate sensors and (red) wind speed sensors.

Figure 7 :Figure 8 :Figure 9 :Figure 10 :

 78910 Figure 7: Solar chimney SCV1 showing the experimental campaign during (a) ventilation only and (b) the charging phase of the experimental protocol without PCM

Figure 11 :

 11 Figure 11: Instrumentation employed for the SCV2 displaying (green) surface temperature sensors on the chimney surface grid, (red) air temperature sensors, (blue) Heat flux plate sensors and (orange) wind speed sensors.

Figure 12 :

 12 Figure 12: Experimental protocol employed for both prototypes picturing the activation of ventilation (open/closed outlet) in blue and solar radiation (on/off halogen lamps)

Figure 13 :

 13 Figure 13: Nomenclature of results for both (a) SCV1 and (b) SCV2.

Figure 14 :Figure 15 :Figure 16 :Figure 17 :

 14151617 Figure 14: C5NoPCM-1 results showing (a) surface temperature and (b) experimental conditions.

Figure 18 :Figure 19 :

 1819 Figure 18: P2C1NoPCM-2 results of (a) surface temperature and (b) operating conditions (external and volume temperature). No insulation and cooler temperature set to 18 • C.

Figure 20 :Figure 21 :Figure 22 :Figure 23 :

 20212223 Figure 20: P2C4NoPCM-3 results of (a) surface temperature and (b) operating conditions (external and volume temperature). With insulation and no cooler temperature support.

Figure 24 :Figure 25 :Figure 26 :

 242526 Figure 24: Air temperature distribution showing inlet level, mid level (1.60 m) and outlet level (3.20 m) under similar operating conditions for (a) P2C1NoPCM-2 and (b) P2C1PCM-2.

Figure 27 :Figure 28 :

 2728 Figure 27: Mass flow rate results under similar operating conditions for C5NoPCM-1 and C1PCM-1.

Figure 29 :Figure 30 :

 2930 Figure 29: Mass flow rate results under similar operating conditions for P2C1NoPCM-2 and P2C1PCM-2.

Figure 31 :

 31 Figure 31: Mass flow rate results under similar operating conditions for P2C4NoPCM-3 and P2C5PCM-2.

Table 1 :

 1 Solar chimney V1.0 dimensions

	Volume	Height [m]	Length [m]	Width [m]
	Control Volume	2.27	1.86	1.00
	Solar Chimney	4.00	0.45	0.50

Table 2 :

 2 Solar chimney V2.0 dimensions

	Volume	Height [m]	Length [m]	Width [m]
	Control Volume	2.14	2.06	1.00
	Solar Chimney	3.50	1.00	0.30

Table 3 :

 3 Technical

	Congealing Range	44 -40	[ • C]
	Heat Storage Capacity ±7%	250 *	[ kJ kg ]
	Specific Heat Capacity	2	[ kJ kgK ]
	Density (Liquid/Solid)	700/800	[ kg m

data of the Rubitherm RT44 PCM and panel characteristics RT44 PCM Properties and characteristics Melting Range 41 -44 [ • C]

Table 4 :

 4 National Instruments acquisition modules description

	Module	Description	Function
		4-Way, 16-Bit,
	NI 9215	Simultaneous Input, 100 kS/s/channel, Module Input	Acquisition of 2 heat flux plate sensors
		Voltage ±10V
		Temperature input	Acquisition of 15
	NI 9217	module for RTD PT100, 4-way,	PT100 surface temperature
		24-Bit	sensors
		16 channels of ±20
		mA input with	Acquisition of 2
	NI 9208	built-in 50/60 Hz	DeltaOhm wind
		rejection for noise	speed sensors
		rejection.
		4-way relay output	Control of lamp
	NI 9481	module, SPST relay, 60 Vdc (1 A)	(on/off) and outlet states
		/ 250 Vrms (2 A)	(open/closed)

Table 5 :

 5 Cycles discussed throughout this section for SCV1 and SCV2

	Cycle	Operating Conditions	Additional Information
	C5NoPCM-1	GHB set inlet temperature	Laboratory with HVAC system
	C7NoPCM-2	GHB set inlet temperature	Laboratory without HVAC system
	C1PCM-1	GHB set inlet temperature	Laboratory with HVAC
	P2C1NoPCM-	GHB set inlet	Extra
	2	temperature	insulation layer
	P2C3NoPCM-	GHB set inlet	Extra
	2	temperature	insulation layer
	P2C4NoPCM-	External	Extra
	3	temperature	insulation layer
	P2C1PCM-2	GHB set inlet temperature	No insulation
	P2C2PCM-2	GHB set inlet temperature	Extra insulation layer
	P2C5PCM-2	External temperature	Extra insulation layer

Acknowledgements

This research was financed by the Mexican organism CONACYT (Consejo Nacional de Ciencia y Tec-20 nología). We thank both CONACYT and the ENTPE for supporting and encouraging this research.


Mohamed Hadi

Mohamed Hadi Abdelhamid

Camille Esquerre-Lamare
email: camille_lamare@hotmail.com

Marie Walschaerts
email: walschaertsm@gmail.com

Gulfam Ahmad
email: gulfam.ahmed@sydney.edu.au

Roger Mieusset
email: mieusset.r@chu-toulouse.fr

Safouane Hamdi
email: hamdi.s@chu-toulouse.fr

Louis Bujan
email: bujan.l@chu-toulouse.fr

Camille Esquerre

Experimental mild increase in testicular temperature has drastic, but reversible, effect on sperm aneuploidy in men: A pilot study

Keywords: Sperm aneuploidy, spermatogenesis alteration, testis temperature increase, male infertility, Fluorescence In Situ Hybridization (FISH)

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

Abstract

In mammals testicular and epididymal temperature increase impairs spermatogenesis. This experimental study investigates the effects of a mild testis temperature increase (i.e. testis temperature remains below core body temperature) on sperm aneuploidy in men. In 5 fertile volunteers a testicular temperature increase was induced by maintaining the testes at suprascrotal position using specially designed underwear for 15 ± 1 hours daily for 120 consecutive days. After heating men were followed for next 180 days. A control group (27 men) was recruited. Semen samples were collected before, during and after heating period and analyzed for chromosomes X, Y and 18 for aneuploidy using FISH. A total of 234,038 spermatozoa were studied by FISH. At day 34 of heating, mean sperm aneuploidy values were not modified. From day 34 of heating until day 45 post heating, FISH evaluation was not possible due to the drastic fall of sperm count. At day 45 post-heating total sperm aneuploidy percentage was twice higher than before heating whereas. Sex disomy (sperm XY18), sex chromosome nullisomy (sperm 18) were significantly higher than controls. These effects were completely reversed at 180 days post heat exposure. Conclusion: A mild rise in testicular temperature significantly increases sperm aneuploidies, reflecting an effect on the meiosis stage of spermatogenesis. The effect of heating was reversible and suggests that recovery of aneuploidy to

Introduction

The adverse effects of increased testicular temperature on spermatogenesis in mammals have been investigated and established for many years. In most mammals, testes are located in the scrotum outside the body, which provides anatomical location that offers lower testicular temperature than core body. In a thermoneutral environment, the scrotal testes are appreciably cooler than the body, but this does not mean that they are kept at a constantly lower temperature [START_REF] Setchell | The parkes lecture heat and the testis[END_REF]. In man as in most other mammals, optimal spermatogenesis requires 2-6°C lower testicular temperature than body and any rise above the physiological temperature of the testes has adverse impact on spermatogenesis [START_REF] Liu | Temperature control of spermatogenesis and prospect of male contraception[END_REF][START_REF] Mieusset | Testicular heating and its possible contributions to male infertility: a review[END_REF].

Several pathological conditions are associated with an increase in testicular temperature which include cryptorchidism [START_REF] Mieusset | Increase in testicular temperature in case of cryptorchidism in boys[END_REF], varicocele [START_REF] Agger | Scrotal and testicular temperature: its relation to sperm count before and after operation for varicocele[END_REF][START_REF] Lund | Varicocele testis and testicular temperature[END_REF][START_REF] Wright | Reduction in testicular temperature after varicocelectomy in infertile men[END_REF] and febrile diseases [START_REF] Evenson | Characteristics of human sperm chromatin structure following an episode of influenza and high fever: a case study[END_REF][START_REF] Sergerie | High risk of temporary alteration of semen parameters after recent acute febrile illness[END_REF]. Exogenous factors such as occupational exposure to high temperatures [START_REF] Bonde | Semen quality in welders exposed to radiant heat[END_REF][START_REF] Hjollund | Impact of diurnal scrotal temperature on semen quality[END_REF], car driving [START_REF] Bujan | Increase in scrotal temperature in car drivers[END_REF] or sauna exposure [START_REF] Garolla | Seminal and molecular evidence that sauna exposure affects human spermatogenesis[END_REF][START_REF] Jockenhovel | A portable digital data recorder for long-term monitoring of scrotal temperatures[END_REF][START_REF] Saikhun | Effects of sauna on sperm movement characteristics of normal men measured by computer-assisted sperm analysis[END_REF] also increase the testicular temperature. Notably, some living conditions such as wearing tight clothing [START_REF] Rock | Effect of induced intrascrotal hyperthermia on testicular function in man[END_REF] or sitting and sleeping postures [START_REF] Hjollund | Diurnal scrotal skin temperature and semen quality[END_REF] are also associated with an increase in testicular temperature which remains in the physiological range [START_REF] Mieusset | Effect of posture and clothing on scrotal temperature in fertile men[END_REF].

The relation between lifestyle factors or exposure to environmental risk factors and sperm aneuploidy was investigated in several studies [START_REF] Jurewicz | The relationship between exposure to air pollution and sperm disomy[END_REF][START_REF] Robbins | Effect of lifestyle exposures on sperm aneuploidy[END_REF][START_REF] Shi | Cigarette smoking and aneuploidy in human sperm[END_REF][START_REF] Young | Environmental exposure to pyrethroids and sperm sex chromosome disomy: a cross-sectional study[END_REF], but to our knowledge, only one epidemiological study has reported the possible link between circumstances known to improve testicular temperature, such as sauna use or type of underwear, and sperm aneuploidy and have shown decrease in sperm aneuploidy in men wearing boxer shorts compared to those wearing tight underwear [START_REF] Jurewicz | Lifestyle factors and sperm aneuploidy[END_REF].

In man, different experimentally methods increasing testicular temperature resulted in a drastic, but reversible, inhibition of spermatogenesis with decrease in sperm count [START_REF] Garolla | Seminal and molecular evidence that sauna exposure affects human spermatogenesis[END_REF][START_REF] Ahmad | Mild induced testicular and epididymal hyperthermia alters sperm chromatin integrity in men[END_REF][START_REF] Mieusset | Hyperthermia and human spermatogenesis: enhancement of the inhibitory effect obtained by artificial cryptorchidism[END_REF][START_REF] Mieusset | Inhibiting effect of artificial cryptorchidism on spermatogenesis[END_REF][START_REF] Rao | Effect of transient scrotal hyperthermia on sperm parameters, seminal plasma biochemical markers, and oxidative stress in men[END_REF][START_REF] Robinson | Intrascrotal hyperthermia induced by scrotal insulation: Effect on spermatogenesis[END_REF] sperm motility [START_REF] Garolla | Seminal and molecular evidence that sauna exposure affects human spermatogenesis[END_REF][START_REF] Ahmad | Mild induced testicular and epididymal hyperthermia alters sperm chromatin integrity in men[END_REF][START_REF] Mieusset | Hyperthermia and human spermatogenesis: enhancement of the inhibitory effect obtained by artificial cryptorchidism[END_REF][START_REF] Mieusset | Inhibiting effect of artificial cryptorchidism on spermatogenesis[END_REF][START_REF] Rao | Effect of transient scrotal hyperthermia on sperm parameters, seminal plasma biochemical markers, and oxidative stress in men[END_REF] and sperm normal morphology [START_REF] Garolla | Seminal and molecular evidence that sauna exposure affects human spermatogenesis[END_REF][START_REF] Mieusset | Hyperthermia and human spermatogenesis: enhancement of the inhibitory effect obtained by artificial cryptorchidism[END_REF][START_REF] Mieusset | Inhibiting effect of artificial cryptorchidism on spermatogenesis[END_REF]. In addition to conventional sperm parameters (count, motility, morphology) the adverse effects of testicular hyperthermia on sperm DNA damage have also been reported [START_REF] Garolla | Seminal and molecular evidence that sauna exposure affects human spermatogenesis[END_REF][START_REF] Ahmad | Mild induced testicular and epididymal hyperthermia alters sperm chromatin integrity in men[END_REF].

It has been suggested that a modification of testicular temperature could be used in two ways: increasing testes temperature to inhibit spermatogenesis or decreasing it to improve spermatogenesis in infertile men who have abnormally elevated testes temperatures. Experimental studies [START_REF] Mieusset | The potential of mild testicular heating as a safe, effective and reversible contraceptive method for men[END_REF][START_REF] Shafik | Testicular suspension as a method of male contraception: technique and results[END_REF][START_REF] Shafik | Contraceptive efficacy of polyester-induced azoospermia in normal men[END_REF] and reviews [START_REF] Anderson | Male contraception[END_REF][START_REF] Setchell | Contraception: Possible physiological bases for contraceptive techniques in the male[END_REF][START_REF] Soufir | Hormonal, chemical and thermal inhibition of spermatogenesis: contribution of French teams to international data with the aim of developing male contraception in France[END_REF] have reported that a mild testis temperature increase could be used as a male contraceptive approach. On the other hand, reducing the testicular temperature using application of an ice bag for 30 min per day for 14 days improves spermatogenesis outcome [START_REF] Robinson | Control of human spermatogenesis intrascrotal temperature[END_REF]. Various devices have been used to decrease testicular temperature and improve spermatogenesis, either in varicocele or in oligozoospermic patients having higher scrotal temperatures [START_REF] Jung | Improvement of semen quality by nocturnal scrotal cooling and moderate behavioural change to reduce genital heat stress in men with oligoasthenoteratozoospermia[END_REF][START_REF] Zorgniotti | Further clinical experience with testis hypothermia for infertility due to poor semen[END_REF][START_REF] Zorgniotti | Scrotal Hypothermia: New theraphy for poor semen[END_REF] . In addition, avoidance of exposure to heat has been reported to prevent infertility [START_REF] Hendry | Investigation and treatment of the subfertile male[END_REF][START_REF] Jung | Influence of the type of undertrousers and physical activity on scrotal temperature[END_REF][START_REF] Lynch | Improved seminal characteristics in infertile men after a conservative treatment regimen based on the avoidance of testicular hyperthermia[END_REF].

We have previously demonstrated that a mild increase in testicular and epididymal temperature (+2°C, i.e. a testicular temperature below core body temperature), induced by maintaining the testes in a suprascrotal position, had drastic effects not only on sperm output and quality but also on sperm DNA fragmentation in humans [START_REF] Ahmad | Mild induced testicular and epididymal hyperthermia alters sperm chromatin integrity in men[END_REF].

While a study in mice [START_REF] Waldbieser | X Y chromosome univalency in the testes of hyperthermic mice: I. Concomitant formation of multinucleated giant cells[END_REF] reported X-Y dissociation increase in spermatocytes from animals that were exposed to an environmental temperature of 35°C for 2-5 days (whole body exposure), to date no study has investigated the effect of a mild testicular temperature increase (testicular temperature below core temperature) on sperm aneuploidy in men. A recent study in man reported increased sperm aneuploidy after testicular exposure to 43°C for two successive days per week for 3 months. [START_REF] Zhang | Impact of a mild scrotal heating on sperm chromosomal abnormality, acrosin activity and seminal alpha glucosidase in human fertile males[END_REF]. Noteworthy, 43°C is very high temperature compared to physiological temperature of testes (≈34.5°C) and sperm aneuploidy is more likely to occur at this higher temperature threshold.

Sperm aneuploidy can be a risk to the embryonic and fetal development and the offspring [START_REF] Templado | New insights on the origin and relevance of aneuploidy in human spermatozoa[END_REF][START_REF] Templado | Aneuploidy in human spermatozoa[END_REF]. The majority of the aneuploidy embryos are not viable resulting into miscarriages but several chromosomal abnormalities, such as autosomal trisomy or sex chromosome aneuploidy, could be non-lethal and compatible with survival resulting in birth and developmental defects [START_REF] Templado | New insights on the origin and relevance of aneuploidy in human spermatozoa[END_REF][START_REF] Templado | Aneuploidy in human spermatozoa[END_REF][START_REF] Eskenazi | Sperm aneuploidy in fathers of children with paternally and maternally inherited Klinefelter syndrome[END_REF][START_REF] Hassold | To err (meiotically) is human: the genesis of human aneuploidy[END_REF].

In this context, the objective of this study was to evaluate the effects of a mild testicular temperature increase (+2°C) on sperm aneuploidy in fertile men and to examine its potential reversibility.

Materials and methods

Study Design

The present study is the second part of a study of the effects of mild induced testes temperature increase on gamete quality [START_REF] Ahmad | Mild induced testicular and epididymal hyperthermia alters sperm chromatin integrity in men[END_REF]. We used the biological specimens that had been frozen during the first part of the study.

Study Population

The study was approved by the Ethics Committee (Comité de Protection des Personnes Sud-Ouest et Outre Mer I) and the protocol has been previously described [START_REF] Ahmad | Mild induced testicular and epididymal hyperthermia alters sperm chromatin integrity in men[END_REF]. The volunteers were recruited through advertisement in the press and local hospital communication portal. A total of 34 men volunteered for the study. After exclusion criteria and financial limitations 6 volunteers (aged 25-35 years) were selected. Only the men who had fathered at least one child, had a normal clinical andrological examination, no current pathology, medical or surgical history, had either no, moderate or occasional alcohol intake were included. None of the participating volunteer was exposed to toxic agents and had no particular professional risk to fertility. One volunteer smoke 20 cigarettes a day in experimental group and 7 men smoke more than 10 cigarettes a day in control group.

One volunteer dropped the study at day 73 of heating due to personal reasons hence the data of this volunteer were excluded. Remaining five volunteers followed the instructions and the protocol and continued until the completion of study. None of the volunteer reported any discomfort during the entire period of study. The aneuploidy results of 27 fertile men were used as control values to compare with aneuploidy values of experimental volunteers (n=5).

Technique for Increasing Testicular Temperature

Increased testicular and epididymal temperature was induced by maintaining the testes in a suprascrotal position by means of specially designed underwear worn 10 15 ± 1 hours daily [START_REF] Mieusset | Testicular heating and its possible contributions to male infertility: a review[END_REF][START_REF] Ahmad | Mild induced testicular and epididymal hyperthermia alters sperm chromatin integrity in men[END_REF][START_REF] Mieusset | The potential of mild testicular heating as a safe, effective and reversible contraceptive method for men[END_REF] for 120 consecutive days. The method was developed by our group and has been tested safe, reversible and reproducible.

Before, commencement of heating participating volunteers of experimental group were trained on how to put the underwear by lifting the testes up at the root of the penis during waking hours. The support to the testes keeping them at suprascrotal position was ensured with the underwear provided with an orifice allowing the scrotum and penis to be exteriorized specifically designed for each volunteer as we previously described [START_REF] Mieusset | Hyperthermia and human spermatogenesis: enhancement of the inhibitory effect obtained by artificial cryptorchidism[END_REF]. The suprascrotal position of the testes results in 1.5 -2°C increase in testicular temperature. Notably, the testicular temperature remains below core human body temperature in this heating method [START_REF] Shafik | Testicular suspension as a method of male contraception: technique and results[END_REF][START_REF] Kitayama | Study on testicular temperature in men[END_REF].

Semen analysis

Semen samples were collected, after mean sexual abstinence of 4.1 (±1.0) days, at specific times according to spermatogenesis chronology as we previously described [START_REF] Jurewicz | The relationship between exposure to air pollution and sperm disomy[END_REF]. After liquefaction at 37°C for minimum of 30 minutes, semen analysis was done according to WHO guidelines [START_REF] Who | WHO laboratory manual for the examination and processing of human semen[END_REF] on an aliquot (200 µL) by a trained technician. The characteristics considered were sexual abstinence (days), ejaculate volume (ml), sperm concentration (SC, 10 6 spermatozoa/ml) and total sperm count (volume × SC = TSC, 10 6 spermatozoa/ejaculate). Total sperm count was the only sperm parameter presented, as it is the true reflect of sperm production.

After initial analysis a part of each sample was frozen in liquid nitrogen in the Germethèque Biobank (BB-0033-00081, France) using routine freezing method currently practiced in our laboratory [START_REF] Esquerré-Lamare | Sperm aneuploidy and DNA fragmentation in unexplained recurrent pregnancy loss: a multicenter case-control study[END_REF] and stored until FISH analysis.

Sperm Fluorescence in situ Hybridization (FISH)

FISH analysis was performed at four time points i.e. before heating at D0, D34, post heating at PH45 and PH180 (Fig. 1). Between day 34 of heating and day 45 post heating total sperm count was drastically reduced due to heating method and did not recover sufficiently until day 45 post heating which rendered the FISH analysis impossible (Figure 1). Frozen cells were thawed and washed twice with 5 ml phosphate buffer saline (PBS) and centrifugation performed at 630g for 10 minutes. Samples were then fixed with fixation solution (acetic acid and methyl alcohol) for 30 min at 4°C. After centrifugation at 1500g for 10 minutes, the supernatant was discarded and the pellet re-suspended. A volume of 10 µl was dropped on a slide and cell density was verified by microscopy and adjusted accordingly. Slides were incubated for a minimum of 2h at -20°C. DNA was decondensed by incubating slides in 1M NaOH for 1 min, washed twice in saline sodium citrate (SSC) buffer, then dehydrated in 70%, 90% and 100% ethyl alcohol baths (2 min each). Each slide was then incubated overnight with the different probes at 37°C (Vysis probes (Abbott), CEP X spectrum green, CEP Y spectrum orange and CEP 18 spectrum aqua). After a 2 min wash in 2SSC 0.4% NP40 at 73°C followed by a 1 min wash in 2SSC 03% NP40, slides were incubated with 1/2000e Hoechst for 3 min and washed for 3 min in PBS. Slides were finally mounted with Antifade mounting medium (Promega, Germany) and stocked at -20°C until reading. Slides were analyzed under a Leica DM 6000 B microscope system.

At each time, a mean of 5149 ± 218 and 5232 ± 190 cells, per subject, were read respectively in experimental group and control group.

Statistical Analysis

Data are presented in the Tables as median and interquartile range Q1-Q3 due to the number of patients and as mean and standard deviation in graphic representations.

Sperm aneuploidy data were compared before, during and after mild induced increase in testicular and epididymal temperature by the Wilcoxon signed ranksum test. Due to the small number of men included in the experimental protocol, we also carried out statistical comparison with a control group of 27 fertile men (a single evaluation of sperm aneuploidy for each man) using the non-parametric Mann-Whitney test. As there were multiple comparisons, a Bonferroni correction was used. P-values of 1.66% were considered significant for the Wilcoxon test and of 1.25% for the Mann-Whitney test.

Statistical analyses were performed using SAS software (9.3, SAS Institute). A pvalue of 5% was considered significant without Bonferroni correction.

Results

The number of volunteers (n=5) was constant during the three study periods (before, during and after heating). Due to technical problems, at D0 and PH45 four volunteers underwent FISH analyses instead of five.

Total sperm count decreased significantly as early as day 34 during heat exposure (Fig. 1) and remained persistently low (azoospermia or severe oligospermia) throughout the heating period (days 0-120). FISH analysis was not possible until day 45 after cessation of heating (PH45).

A total of 234,038 spermatozoa were analyzed by FISH. Before commencement of experimental heating (Day 0), no significant differences were found for aneuploidy percentages between the control group (n = 27) and the five volunteers except for median frequencies of XXY18. However, the range of frequencies difference was very small, ranging from 0.00 to 0.02 (Table 1).

Table 1 represents sperm aneuploidy percentages at the different time points of the study. The major significant results were an increased total aneuploidy, sex disomy (sperm XY18) and sex chromosome nullisomy (sperm 18) at day 45 post heating (PH45). The percentages of sperm with sex disomy and sex nullisomy were two and three times more elevated, respectively, than before heating period (D0). Percentage of total aneuploidy was twofold higher at PH45 than before heating (D0) (Table 1, Fig. 2).

At day 180 post heating (PH180), sperm aneuploidy percentages did not differ from the values before heating (Fig. 2), which suggests that the effects of heating were reversible.

In this study, we defined an abnormal aneuploidy rate as a rate above the 90 th percentile of control group values. This enabled us to calculate the number of volunteers who had an abnormal aneuploidy rate at different time points.

The results indicate that at 45 post heating, all volunteers had a higher number of abnormal sperm with sex disomy XY18, sex nullisomy and total sperm aneuploidy (Fig. 3) which did not recover to values before heating period until day 180 post heating suggesting that minimum of two cycles of spermatogenesis and epididymal transit after discontinuation of heating are required in order to 255 restore normal spermatogenesis with no risk of aneuploidy (Fig. 3). 256

Discussion

The negative impact on spermatogenesis after testicular exposure to a mild or high temperatures increase has been demonstrated by numerous experimental human and animal studies. A recent study reported an increases sperm aneuploidy rate when testes were exposed to 43°C [START_REF] Zhang | Impact of a mild scrotal heating on sperm chromosomal abnormality, acrosin activity and seminal alpha glucosidase in human fertile males[END_REF]. However, these findings were taken at one-point time and reversibility was not evaluated. Moreover, 43°C is a pathological temperature which is much higher than testes and core body temperature and sperm aneuploidies are more likely to occur at this high threshold. No study has investigated the effect of a mild increase in testicular temperature (<37°C) on the frequency of sperm chromosomal abnormalities.

In present study by inducing a mild increase in testes and epididymal temperature for 15 ± 1 hours daily for 120 consecutive days, we show that this temperature threshold is sufficient to increase sperm aneuploidy in men and that this effect is reversible after cessation of heating which suggests that testicular heating is the cause of sperm aneuploidy.

At day 34 of heating, the percentage of chromosomal abnormalities were not affected by the mild temperature increase. In accordance with the physiology of the spermatogenesis process and epididymal transit timing, spermatozoa collected at D34 were at the late spermatocyte stage (end of meiosis) or early round spermatids in the testes when heat was induced: i.e. they were subjected to heat but were released from progenitor germ cells that were not exposed to heating. This is in agreement with previous reports indicating that most chromosomal abnormalities in sperm result from meiotic errors during the spermatogenesis process [START_REF] Griffiths | Chromosome mutation II: Changes in chromosome number[END_REF][START_REF] Uroz | Meiotic non-disjunction mechanisms in human fertile males[END_REF].

At day 45 post heating (PH45) we found a significant increase of disomic sperm (XY18 sperm: non-disjunction of the sexual chromosome at meiosis I) or nullisomic sperm [START_REF] Mieusset | Effect of posture and clothing on scrotal temperature in fertile men[END_REF] and a slight increase of diploidy sperm (XY1818), consequently total aneuploidy increased at this time. It is noteworthy that spermatozoa collected at day 45 after heating were more probably affected by heating at the meiosis stage, according to spermatogenesis and epididymis physiology as previously published [START_REF] Ahmad | Mild induced testicular and epididymal hyperthermia alters sperm chromatin integrity in men[END_REF].

The effects of a mild increase in testes temperature are reversible, as indicated by recovery of aneuploidy values to pre-heating values at PH180. The time require to recover the aneuploidy to control values suggest that reversibility requires more than two cycles of spermatogenesis.

A recently published study examined sperm aneuploidy in 10 men before and during scrotal heating using an electric warming bag to increase the testes temperature to 43°C for 30-40 min two days per week for 3 months [START_REF] Zhang | Impact of a mild scrotal heating on sperm chromosomal abnormality, acrosin activity and seminal alpha glucosidase in human fertile males[END_REF].

Compared to pre-heating values, a significant increase in the sum rate of sperm chromosomal 13, 18, 21, and sum of X and Y sperm was reported at the end of the 3 months of heating. In this last study, the increase of sum rate of aneuploidy for X and Y at the end of the 3-month period of heating was 14.2-fold higher than before heating values. This is much higher than the 2 to 3-fold increase we observed in the present study where temperature increase was very mild.

However, the aneuploidy values after cessation of heating were not reported in the study by Zhang et al [START_REF] Zhang | Impact of a mild scrotal heating on sperm chromosomal abnormality, acrosin activity and seminal alpha glucosidase in human fertile males[END_REF] which lacks information about the reversibility.

Further, only 200 sperm cells were analyzed by Zhang et al [43[ compared to present study which report analysis of 5149 ± 218 and 5232 ± 190 cells in experimental volunteers and controls respectively. Relation of lifestyle factors, such clothing, with sperm aneuploidy has also been reported. Jurewicz et al [START_REF] Jurewicz | Lifestyle factors and sperm aneuploidy[END_REF] reported that use of boxer short decreases sperm aneuploidy rate compared to tight underwear.

In animal models, only three studies have reported the effect of induced increased temperatures on germ cell chromosomal abnormalities and abnormal chromosome configurations [START_REF] Waldbieser | X Y chromosome univalency in the testes of hyperthermic mice: I. Concomitant formation of multinucleated giant cells[END_REF]. Exposing mice to an environment of approximately 35 ± 1°C and 65% relative humidity for 2-5 days led to significant peaks of X-Y dissociation 5 days after the end of heat stress which recovered to control levels 50-70 days after cessation of heat stress (≈1.5 to 2 cycles of spermatogenesis in mice).

In another study [START_REF] Van Zelst | XY chromosome dissociation in mice and rats exposed to increased testicular or environmental temperatures[END_REF], testes of mice and rats under anesthesia were heated in a water bath at 42°C for 20 min which resulted in an increase in the percentage of X-Y univalents after 6 days of heat exposure in mice and 12 days in rats. These results suggest an action of heating in pre-meiotic or early prophase I stages of spermatogenesis, which was also suggested by Garriott and Chrisman [START_REF] Garriott | Hyperthermia induced dissociation of the X Y bivalent in mice[END_REF].

The precise mechanism of aneuploidy induction in man following mild testis temperature increase is not known. However, meiosis is a structural dynamic process, broadly conserved across eukaryotes, and temperature appears to be a major challenge for chromosome segregation during the meiosis stage.

Temperature increase was associated with failure of synapsis and reduced chiasma frequency in plant and animals at both high and low temperatures [START_REF] Bomblies | Meiosis evolves: adaptation to external and internal environments[END_REF].

Heat shock proteins (HSPs) play a protective role in response to cell stress and several HSPs were induced mainly in spermatocytes and round spermatids when testis temperature was increased [START_REF] Widlak | The role of heat shock factors in mammalian spermatogenesis[END_REF]. The HSP70 gene is expressed during the meiosis prophase, and HSP70 protein is present in the synaptonemal complex and in attachment plaques at the ends of chromosomes. Desynapsis is disrupted in male mice lacking this protein [START_REF] Eddy | Role of heat shock protein HSP70-2 in spermatogenesis[END_REF][START_REF] North | Reversible meiotic abnormalities in azoospermic men with bilateral varicocele after microsurgical correction[END_REF]. Moreover, heat stress following experimental cryptorchidism in rats resulted in modifications in the expression of 594 genes in pachytene spermatocytes [START_REF] Yadav | The thermo-sensitive gene expression signatures of spermatogenesis[END_REF]. Some cases of varicoceles are associated with abnormally elevated testes temperature. It is noteworthy that in patients with varicocele, the frequency of meiotic abnormalities such as partial asynapsis, chromosome breaks and a high rate of sex vesicle-autosome association were higher than in a control group [START_REF] North | Reversible meiotic abnormalities in azoospermic men with bilateral varicocele after microsurgical correction[END_REF].

Further analysis after varicocele surgery shoed now abnormalities suggesting that the meiotic alterations were due to increased temperature and were reversible.

Likewise, a comparison of sperm aneuploidy frequency before and after varicocelectomy showed decrease, though not significant, in the sperm aneuploidy frequency [START_REF] Acar | Comparison of semen profile and frequency of chromosome aneuploidies in sperm nuclei of patients with varicocele before and after varicocelectomy[END_REF].

One of the limitations of our study is the small number of volunteers included in this experimental protocol. The other limitation is the small number of chromosomes studied by FISH (X, Y, 18). However, the inter-chromosomal effect has been reported using CGH array [START_REF] Hornak | Comprehensive meiotic segregation analysis of a 4-breakpoint t (1; 3; 6) complex chromosome rearrangement using single sperm array comparative genomic hybridization and FISH[END_REF][START_REF] Patassini | Molecular karyotyping of human single sperm by array-comparative genomic hybridization[END_REF] and it has been suggested that the results obtained by studying one set of chromosomes could be extended to others [START_REF] Frias | NOVP chemotherapy for Hodgkins disease transiently induces sperm aneuploidies associated with the major clinical aneuploidy syndromes involving chromosomes X, Y, 18, and 21[END_REF]. The absence of longitudinal follow-up of the control group could be another limitation of our study. Testicular temperature was not measured in this study owing to invasive approach and was based on the existing literature [START_REF] Shafik | Testicular suspension as a method of male contraception: technique and results[END_REF][START_REF] Kitayama | Study on testicular temperature in men[END_REF].

While further studies are needed to clarify the relationship between sperm aneuploidy and various circumstances resulting in testes temperature increase, findings of current study may have clinical implications. In infertile men, it seems reasonable to avoid or treat situations (fever, varicocele, ascending testis or mispositioning of testis, and occupational or professional exposure) which can result in increased scrotal or testicular temperature. If such circumstances are unavoidable, FISH analysis of sperm may be advised to identify aneuploidy which can pose possible risk to the future embryo.

In conclusion, this study shows that a mild induced increase (≈2°C) in testicular and epididymal temperature (15 ± 1 hour daily for 120 consecutive days) results in increased sperm aneuploidy in men, which reflects an effect of temperature on the meiosis stage of spermatogenesis. This effect was reversible and suggests waiting period of minimum 2 cycles of spermatogenesis if a couple is planning to conceive allowing cells sufficient time to recover from heat stress. Nevertheless, the current results are based on a smaller population which does warrant validation of these findings on larger cohort of men. Sperm aneuploidies (%) before, during and after a mild increase in testicular and epididymal temperature in men. Sperm chromosomal abnormalities (%) measured at the four time points: before heating (D0), during heating (H34), and after heating (PH45 and PH180). Means ± SEM are represented. Mild induced testicular and epididymal heating was discontinued at D120.

Figure 3

Number of volunteers whose percentage of sperm aneuploidies before, during and after a mild induced increase in testicular and epididymal temperature was greater than the 90e percentile of the control group.

Figure 1

 1 Figure 1

Figure 2

 2 Figure 2

This study was supported by a grant from CHU de Toulouse N° 09 161 02. to thank Nina Crowte for text editing.

Conflicts of Interest:

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported * p <0.0125 between controls and volunteers at time 0, H34, PH [START_REF] Templado | Aneuploidy in human spermatozoa[END_REF]