Abstract
As human space exploration extends toward long-duration missions and off-Earth settlements, understanding the impact of microgravity on reproduction is vital for sustaining life beyond Earth. This study investigates how simulated microgravity influences sperm navigation, fertilization, and subsequent early embryo development across three mammalian species; human, mouse, and pig, using a dual-axis 3D clinostat and clinically relevant in vitro culture systems. Sperm function was assessed via microchannels, revealing that microgravity impaired directional navigation and fertilization capacity in a time- and species-dependent manner. Fertilization still occurred, indicating compensatory mechanisms within subsets of sperm. Notably, progesterone partially restored navigation in human sperm under microgravity, suggesting the potential of chemical cues to mitigate gravitational absence. In vitro fertilization and embryo culture were performed under clinical assisted reproduction conditions, with time-lapse monitoring and blastocyst lineage characterization. Microgravity exposure during fertilization (4-6 h) compromised blastocyst development in pigs and led to elevated inner cell mass and epiblast counts in pig and mouse embryos. Prolonged exposure (24 h post-fertilization) resulted in developmental delays and reduced blastocyst cell numbers in mice. These findings underscore the resilience and vulnerability of reproductive processes under altered gravitational conditions, emphasizing the critical need to optimize peri-conception environments for successful reproduction for future space missions.
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Introduction
Space exploration continues to inspire scientific advancement, with a renewed focus on long-term missions and planetary settlements. NASA’s ARTEMIS program aims to return humans to the Moon by 2029, while SpaceX anticipates its first crewed Mars missions by 20301,2,3,4. As humanity moves toward permanent off-Earth habitation, understanding how extraterrestrial conditions affect reproduction is essential for crew health and the sustainability of life beyond Earth.
Extraterrestrial environments are characterized by altered gravity, increased radiation, and extreme climates. Earth’s gravity is 1×g, compared with 0.377×g on Mars, 0.166×g on the Moon, and near-zero in the vacuum of space5, with these reduced gravity conditions known as microgravity. Gravity has played a fundamental role in the evolution of life, with cells relying on mechanosensing to convert physical forces into biochemical signals6. Studies of astronauts returning from the international space station (ISS) have shown impacts of a reduced gravity environment on several key physiological systems, including a fluid shift to reduce circulating blood volume, increased muscular atrophy and a 1-2% bone loss every month, increasing ataxia, postural disturbances and fatigue and a reduction in the differentiation and polarisation of immune cells7,8,9,10.
Reproductive function is similarly gravity sensitive. Microgravity has been shown to impair mouse embryonic stem cell differentiation during space flights (i.e. TZ-1 and STS-131)11,12, inhibit human endometrial decidualization13, and affect male sex hormone levels and offspring sex ratios14. Nevertheless, the specific effects of microgravity on gamete function and the earliest stages of embryogenesis remain unclear. One of the earliest investigations, conducted on Cosmos 1887, revealed that space-exposed rats had reduced testicular mass and fewer spermatogonia compared with controls, though findings could not be attributed to microgravity alone15. Subsequent studies assessing sperm function under microgravity have yielded mixed results16. Studies on pre-implantation embryo development are similarly varied. Mouse embryos at the 2-cell stage, sent on the Columbia Space Shuttle (STS-80), failed to develop to the blastocyst stage17, while those aboard China’s SJ-10 satellite did produce blastocysts, albeit of lower quality18. Mouse embryonic stem cells exposed to microgravity had impaired differentiation capacity and reduced expression of specific tissue markers11. However, mouse embryos fertilized in vitro under simulated microgravity successfully reached the blastocyst stage and produced live offspring19,20. Meanwhile, in vitro fertilization under simulated microgravity resulted in developmental arrest at the 8-cell stage in cattle21.
The biological effects of microgravity remain unclear, partly due to challenges in isolating gravitational effects from other variables such as radiation, culture conditions, and sample handling. Various microgravity simulation models have been employed, each with limitations. Spaceflight, although highly relevant, cannot differentiate between radiation and microgravity effects and often relies on suboptimal culture systems with delayed assessments18,21. Parabolic flight only allows for ~30 s of microgravity exposure22, while hindlimb suspension models introduce stress-induced confounding variables23. Random positioning machines (RPMs) or 3D clinostats, simulate microgravity by rotating biological samples in all spatial directions, effectively randomizing the gravity vector, and tend to be the gold standard for on Earth microgravity research24,25. However, many studies using these platforms have failed to apply optimal gamete and embryo culture conditions, such as clinical grade gamete and embryo culture media and low oxygen tension19,20, limiting their conclusions on microgravity’s true effect.
As we progress toward interplanetary missions, understanding how microgravity affects the earliest stages of mammalian reproduction is critical. We, therefore, aimed to systematically evaluate the effects of simulated microgravity on sperm function, navigation, and fertilization capacity in three mammalian species (human, mouse, and pig) as well as the downstream consequences of microgravity exposures during fertilisation on pre-implantation embryo development in mouse and pig. Using a dual-axis 3D clinostat, we were able to for the first time simulated microgravity under conditions mimicking the female reproductive tract and clinical assisted reproductive technology (ART) environments. This study addresses existing knowledge gaps through multi-species physiologically relevant models, providing a solid foundation for the future of space reproductive health research.
Results
Validation of gamete and embryo culture model in 3D clinostat
To ensure that our measurements of sperm function, fertilization and early embryo formation were a result of cellular behaviors and not mixing or sheer force occurring due to the clinostat rotation (Supplementary Video 1), several experiments were performed to measure the effect of positioning relative to the center of the axis, diffusion across the channel, effect of viscosity, different testing vessels and liquid volumes, and considerations of the fluid dynamics within our vessels (Supplementary Fig. 1). To overcome issues of sample mixing due to changes in sample viscosity all human semen underwent a simple wash to remove their seminal plasma fraction and resuspended with media prior to addition to the channel. After testing the impact of positioning of the channel slide relative to sperm motility, noting that positioning in outer channels resulted in declines in sperm motility likely due to fluid shearing, all subsequent experiments were exclusively performed using the center wells of the channel slide (Supplementary Fig. 1C–G). Using phenol-stained media and fluorescent beads placed in the channel opening we determined that the rotation of our 3D clinostat was not inducing sample mixing, and that sperm must actively swim through the channel to reach the other side (Supplementary Fig. 1H–I). We also performed control experiments to further show that culturing sperm, oocytes and embryos in the channel did not hinder fertilization or pre-implantation embryo development with outcomes similar with standard culture controls using clinical IVF grade embryo culture dishes (Supplementary Fig. 1J–O).
Microgravity reduces human sperm navigational ability, without altering motility
To assess the effects of simulated microgravity on human sperm navigation and motility, washed spermatozoa were incubated in standard or female tract-mimicking media under normal gravity or simulated microgravity at 5% O₂, 6% CO₂ and 37 °C. Following transit through a microchannel system simulating the female reproductive tract, sperm recovery and motility parameters were quantified using computer-assisted semen analysis (CASA) (Fig. 1A). We observed a significant reduction in the percentage of sperm that could successfully navigate the channel and reach the collection point in both standard culture and the female fluid mimic (P = 0.002 and P = 0.007, Fig. 1B) under simulated microgravity compared with normal gravity conditions. Interestingly, we observed no changes to sperm motility and kinetic parameters, including total motility (Fig. 1C), progressive motility (Fig. 1D), curve linear velocity (Fig. 1E), or lateral head displacement (Fig. 1F), indicating that the decline in recovery was not due to a change in motility.
A Human spermatozoa challenged to swim through a micro-channel slide under microgravity in either gamete or female fluid mimic media prior to sperm functional assessments (compared to normal gravity control); B swim through recovery efficiency; C total motility; D progressive motility; E curve linear velocity F lateral head displacement; G swim through recovery efficiency following exposure to progesterone at 10 and 100 µM; H binding to hyaluronan (normal gravity vs microgravity only); I swim through recovery efficiency following ‘swim up’ sperm selection; and J binding to hyaluronan following ‘swim up’ sperm selection. Data are presented as mean ± SEM, with unique symbols representing individual men. Normal gravity controls denoted in white (gamete medium), and light purple with diagonal stripes (female fluid mimic medium), whilst simulated microgravity treatment indicated by pink (gamete medium) or dark purple with diagonal stripes (female fluid mimic medium). Experiments where sperm were selected by ‘swim up’ are indicated by red. Statistical significance characterized as * P < 0.05, and ** P < 0.01. Parts of this image were created in BioRender. Mcpherson, N. (2026) https://BioRender.com/k8p7vd5.
To ensure the observed effects resulted from simulated microgravity and not an artifact caused by the 3D clinostat, we performed rotational control experiments in standard culture conditions as outlined above (Supplementary Fig. 2A). No differences were seen between the rotational control and normal gravity control for sperm swim through recovery (Supplementary Fig. 2B), total motility (Supplementary Fig. 2C), progressive motility (Supplementary Fig. 2D), or lateral head displacement (Supplementary Fig. 2F), although a 20% decline was noted for curve linear velocity (P = 0.04, Supplementary Fig. 2E), despite no change observed at simulated microgravity (Fig. 1E).
To further understand how simulated microgravity was negatively impacting sperm’s capacity to navigate the channel slide, we next assessed whether this loss of navigational ability could be rescued through the addition of a chemotactic factor, progesterone. Progesterone was chosen as it is secreted by the cumulus-oocyte complex and activates sperm Catsper channels enhancing motility and navigation towards the oocyte26. We found that whilst 10 µM of progesterone did not improve recovery compared with normal gravity (P = 0.04, Fig. 1G), a much higher dose of 100 µM progesterone was able to restore sperm recovery to levels observed in the normal gravity control (Fig. 1G), without any changes to motility (Supplementary Fig. 3A–D). This indicates that the presence of a high dose chemoattractant can overcome the negative effects simulated microgravity has on sperm navigation.
Next, we wanted to further test sperm functional capacity by assessing their ability to bind to hyaluronan, a molecule present in the extracellular matrix of the cumulus-oocyte complex27. This measure is directly correlated with sperm quality, with sperm binding to hyaluronan displaying lower sperm DNA fragmentation levels and improved maturity and clinical outcomes following intracytoplasmic sperm injection (ICSI)28,29,30,31,32. Sperm recovered after simulated microgravity exposure had a greater capacity to bind hyaluronan compared with normal gravity control (P = 0.04, Fig. 1H), suggesting that microgravity could be acting as a positive selective pressure, selecting for sperm with greater affinity to bind an oocyte.
To further test this concept, we performed the same experiment in spermatozoa isolated via swim-up sperm selection, and prior to the channel challenge under simulated microgravity. Similar to washed semen, we observed a significant reduction in the percentage of progressively motile sperm successfully able to navigate the channel under simulated microgravity (P = 0.03, Fig. 1I), without any changes seen to motility (Supplementary Fig. 4A–D). Further, when assessing the ability of these sperm to bind hyaluronan we found that preselecting for motile sperm prior to exposure to simulated microgravity and the channel did not add any additional selective pressures with binding levels similar between groups (Fig. 1J).
Microgravity reduces mouse sperm’s ability to navigate and fertilize an oocyte but increases the number of fetal cells in the blastocyst
To better understand the effects that simulated microgravity has on sperm fertilization capacity and early embryo formation we continued our experiments in the mouse. Firstly, we confirmed that mouse sperm behaved similarly at simulated microgravity to that of human sperm (Fig. 2A). After 1 h of incubation at simulated microgravity, no difference in sperm recovery rate was seen (normal gravity 3.5 ± 2.2% vs microgravity: 2.4 ± 1.4%, Fig. 2B). As very little sperm were recoverable after 1 h (<5%), a 2 h and 4 h time point were assessed. Similar to human sperm, the ability of mouse sperm to navigate a channel slide was significantly reduced following 2 h simulated microgravity compared with normal gravity control (P = 0.02, Fig. 2B), however by 4 h, sperm recovery rates were similar (Fig. 2B). We also observed no differences in total motility (Fig. 2C), progressive motility (Fig. 2D), curve linear velocity (Fig. 2E), or lateral head displacement (Fig. 2F) in the sperm subjected to 2 h of microgravity compared with normal gravity. By 4 h, progressive motility was reduced under simulated microgravity (P = 0.009, Fig. 2D) while total motility (Fig. 2C), curve linear velocity (Fig. 2E) and lateral head displacement (Fig. 2F) were unaffected. We also performed rotational control experiments after 2 h of simulated microgravity and observed no differences in mouse sperm recovery rates or motility kinetics (Supplementary Fig. 5A–F), further highlighting that the observed effects were likely due to simulated microgravity.
A Mouse sperm was collected from the cauda epididymis and vas deferens into G-IVF PLUS while pig sperm was washed free of extender and placed in TALPs fertilization media. Sperm from both species were challenged to traverse the micro-channel slide at either normal gravity or simulated microgravity prior to sperm functional assessments. Mouse sperm (B) swim through recovery efficiency, C total motility, D progressive motility, E curve linear velocity and F lateral head displacement. Pig sperm (G) total motility, H progressive motility, I curve linear velocity J lateral head displacement K representative images of pig sperm agglutination at normal gravity and simulated microgravity and L proportion of pig sperm with low, medium or high levels of agglutination. Data are presented as mean ± SEM, with unique symbols representing individual mice and pigs. Normal gravity controls denoted in white (mouse and pig) and white diagonal stripes (mouse 4 h exposure), and simulated microgravity denoted in light blue (mouse 2 h exposure), dark blue with diagonal stripes (mouse 4 h exposure) and green (pig). Statistical significance characterized as * P < 0.05, and ** P < 0.01. Parts of this image were created in BioRender. Mcpherson, N. (2026) https://BioRender.com/nrkg7g0.
We next investigated the impact that simulated microgravity had on in vitro fertilization (IVF). By placing sperm and cumulus-oocyte complexes (COCs) at opposite ends of the channel we were able to determine the impact microgravity has on sperm fertilization capacity. Following 4 h of coincubation at simulated microgravity, COCs were removed and placed into either a standard bench top culture or timelapse incubator under normal gravity for a further 96 h to determine any downstream effects on pre-implantation embryo quality (Fig. 3A). When the COCs were removed from the channel following IVF, we visually observed a reduction in the numbers of oocytes successfully denuded by sperm at simulated microgravity after 4 h (Fig. 3D). This translated into a 30% reduction in fertilization rates as assessed by 2-cell development 24 h post-insemination (P = 0.03, Fig. 3B). No differences were observed in the percentage of embryos that were on-time ( > expanded blastocyst) at 96 h of culture (Fig. 3C) between those fertilized at simulated microgravity compared with normal gravity control. Although there were no significant differences seen in embryo morphokinetics (Supplementary Fig. 6 and Supplementary Video 2), there was a trend for faster time from PN appearance to hatching blastocyst (P = 0.07, Supplementary Fig. 6F) and 2-cell to hatching blastocyst (P = 0.05, Supplementary Fig. 6J) following fertilization at simulated microgravity.
A Female mice were super ovulated and cumulus oocyte complexes recovered from the oviduct and placed at the end of the micro-channel slide. Mouse sperm was collected from the cauda epididymis and vas deferens and placed at the other end of the micro-channel slide. Sperm were required to traverse the micro-channel slide at either normal gravity or simulated microgravity to fertilize oocytes for 4 h. Following incubation all groups were cultured in standard clinical in vitro embryo culture conditions for a further 96 h for assessments of pre-implantation embryo quality. B Proportion of 2-cells, 24 h post insemination, C proportion of expanded blastocysts, hatching blastocysts and fully hatched blastocyst from 2-cells, 96 h post insemination, D representative images of oocytes following 4 h insemination period at simulated microgravity or normal gravity control and subsequent blastocyst formation 96 h post insemination, E total blastocyst cell number (TCN), 96 h post insemination, F total blastocyst inner cell mass cell (ICM) number (Oct4 positive), 96 h post insemination, G total blastocyst epiblast cell number (Nanog positive), 96 h post insemination, H proportion of blastocyst ICM relative to TCN, I proportion of blastocyst epiblast cells relative to TCN, J proportion of blastocyst epiblast cells relative to ICM and K representative images of blastocyst cell numbers generated from 4 h insemination window at simulated microgravity and normal gravity control. Data are presented as mean ± SEM, with individual culture replicates indicated by unique symbols or individual embryos (circles). N = 82 embryos assessed for fertilisation and blastocyst development and N = 27 for cell numbers from 5 replicates for normal gravity control and N = 156 embryos assessed for fertilisation and blastocyst development and N = 46 for cell numbers from 5 replicates for simulated microgravity. Normal gravity controls denoted in white, and simulated microgravity denoted in light blue. Statistical significance characterized as * P < 0.05, and ** P < 0.01. P-values between 0.05 and 0.1 are presented numerically. Parts of this image were created in BioRender. Mcpherson, N. (2026) https://BioRender.com/kvmm95y.
Blastocysts were then fixed at 96 h and underwent differential staining to determine cell numbers as a marker of embryo quality. We observed no differences in total blastocyst cell numbers, nor blastocyst inner cell mass cell numbers between those embryos fertilized at simulated microgravity compared with normal gravity control (Fig. 3E, F). However, we observed a significant increase in the number of epiblast cells (P = 0.01, Fig. 3G), resulting in an increase in epiblast to total cell number and epiblast to inner cell mass ratio (P = 0.006 and P = 0.04, respectively, Fig. 3I–K) in those embryos fertilized at simulated microgravity. These findings suggest that simulated microgravity may act as a selective pressure, where fewer sperm successfully reach the oocyte, but those that do may possess characteristics associated with higher functional competence. This could contribute to embryos exhibiting increased epiblast cell numbers, which are generally considered indicative of stronger developmental potential33,34,35.
To ensure the effects we observed were a result of simulated microgravity and not a result of being placed on the 3D clinostat, we performed rotational control experiments (Supplementary Fig. 7A). No differences in fertilization, total blastocyst development, embryo morphokinetics or blastocyst cellular allocation/numbers were seen between our rotational control and normal gravity control (Supplementary Fig. 7B–J).
To determine if the decline in fertilization rates following 4 h co-incubation at microgravity was due to the reduced ability of sperm to navigate to a COC, we extended our co-culture period out to 24 h (Fig. 4A). As hypothesized, increasing the co-culture period resulted in similar fertilization rates between simulated microgravity and our normal gravity control (Fig. 4B). However, the time taken to get from 2-cell to 4-cell and 2-cell to 8-cell was increased in embryos exposed to simulated microgravity for the first 24 h (Both P = 0.05, Supplementary Fig. 8B-C and Supplementary Video 3) and there was also a trend for decreased percentage of on-time blastocysts ( > expanded blastocyst) at 96 h of culture under 24 h simulated microgravity (P = 0.09, Fig. 4C, D). Interestingly, when assessing blastocyst cell numbers at 96 h, blastocysts that had been created by sperm, COCs and zygotes exposed to simulated microgravity for the first 24 h had reduced epiblast cell numbers (P = 0.04, Fig. 4G, K) and a trend for reduced total cell number (P = 0.05, Fig. 4E, K) and inner cell mass cell numbers (P = 0.06, Fig. 4F, K) compared with normal gravity control. There was no difference in the distribution of inner cell mass cells and epiblast cells within the blastocyst under simulated microgravity (Fig. 4H–K), indicating that the effects were due to a global reduction in blastocyst cell numbers as supposed to a change in cell linage. So, while increasing the incubation time of sperm and COCs under simulated microgravity restored fertilization rates, the exposure of the developing zygote to microgravity for the first 24 h of development negatively impacted blastocyst quality.
A Female mice were super ovulated and cumulus oocyte complexes recovered from the oviduct and placed at the end of the micro-channel slide. Mouse sperm was collected from the cauda epididymis and vas deferens and placed at the other end of the micro-channel slide. Sperm were required to traverse the micro-channel slide at either normal gravity or simulated microgravity to fertilize oocytes for 24 h. Following incubation all groups were cultured in standard clinical in vitro embryo culture conditions for a further 72 h for assessments of pre-implantation embryo quality. B Proportion of 2-cells, 24 h post insemination, C proportion of expanded blastocysts, hatching blastocysts and fully hatched blastocyst from 2-cells, 96 h post insemination, D representative images of blastocyst formation at 96 h post insemination following a 24 h insemination period at simulated microgravity or normal gravity control, E total blastocyst cell number (TCN), 96 h post insemination, F total blastocyst inner cell mass (ICM) cell number (Oct4 positive), 96 h post insemination, G total blastocyst epiblast cell number (Nanog positive), 96 h post insemination, H proportion of blastocyst ICM relative to TCN, I proportion of blastocyst epiblast cells relative to TCN, J proportion of blastocyst epiblast cells relative to ICM and K representative images of blastocyst cell numbers generated from 24 h insemination window at simulated microgravity and normal gravity control. Data are presented as mean ± SEM, with individual culture replicates indicated by unique symbols or individual embryos (circles). N = 187 embryos assessed for fertilisation and blastocyst development and N = 35 for cell numbers from 6 replicates for normal gravity control and N = 239 embryos assessed for fertilisation and blastocyst development and N = 23 for cell numbers from 6 replicates for simulated microgravity. Normal gravity controls denoted in white, and simulated microgravity denoted in dark blue. Statistical significance characterized as * P < 0.05, and ** P < 0.01. P-values between 0.05 and 0.1 are presented numerically. Parts of this image were created in BioRender. Mcpherson, N. (2026) https://BioRender.com/ix6duwb.
Microgravity does not alter pig sperm motility but does reduce the ability of sperm to agglutinate at 38.5 oC, fertilize an oocyte and create an embryo
To further determine if the results seen in the mouse were reproducible in other species, we assessed fertilization and early embryo development in another mammalian model—the pig. Pig sperm recovery could not accurately be assessed due to the heavy agglutination of sperm within the channel under normal gravity conditions (Fig. 2K). Agglutination of pig sperm is a direct consequence of membrane modifications that occur during the capacitation process36, however, the absence of heavy sperm agglutination under simulated microgravity conditions (Fig. 2K, L) may indicate a change to pig sperm physiology, although this was not further investigated. Similar to human, we observed no differences in total sperm motility, progressive motility, curve linear velocity, or lateral head displacement between those exposed to simulated microgravity compared with normal gravity control (Fig. 2G–J).
We next investigated the impact simulated microgravity had on the ability of pig sperm to fertilize an oocyte (Fig. 5A). After 6 h of sperm and COC co-incubation at simulated microgravity, we collected oocytes and cultured them in standard culture conditions for a further 144 h. Similar to the mouse, we observed a reduction in fertilization rates as assessed by 2-cell development at 56 h post-insemination (P = 0.01, Fig. 5B). In contrast to the mouse, we also observed a reduction in the ability of fertilized embryos to develop to the expanded blastocysts at 144 h (P = 0.005, Fig. 5 C-D). Blastocyst differential staining was also performed at 144 h. Like the mouse, we observed in those embryos that reached the blastocyst no differences in total cell numbers or trophectoderm cell numbers in embryos fertilized at simulated microgravity (Fig. 5E, G). Further, matching the mouse data we also observed a significant increase in the number of inner cell mass cells of blastocysts (P = 0.05, Fig. 5F). This resulted in an increase in inner cell mass cell to total cell number ratio (P = 0.003, Fig. 5H, K) and a decrease in the trophectoderm to total cell number ratio and trophectoderm to inner cell mass ratio (both P = 0.003, Fig. 5I–K) under simulated microgravity. Data from the pig further indicates that sperm exposed to simulated microgravity are less likely to reach and fertilize an oocyte. However, those that do fertilize may exhibit characteristics associated with higher functional competence, as reflected by embryos forming blastocysts with increased allocation of cells to the inner cell mass, which comprises both the primitive endoderm and epiblast cells.
A Pig ovaries were collected from the abattoir and cumulus oocyte complexes (COCs) aspirated and cultured for 44 h to obtain mature COCs. Mature COCs were placed at the end of the micro-channel slide and washed extended boar sperm placed at the other end. Sperm were required to traverse the micro-channel slide at either normal gravity or simulated microgravity to fertilize oocytes for 6 h. Following incubation all groups were cultured in standard clinical in vitro embryo culture conditions for a further 144 h for assessments of pre-implantation embryo quality. B Proportion of 2-cells, 56 h post insemination, C proportion of expanded blastocysts and hatching blastocysts from 2-cells, 144 h post insemination, D representative images of blastocyst formation at 144 h post insemination following a 6 h insemination period at simulated microgravity or normal gravity control, E total blastocyst cell number (TCN), 144 h post insemination, F total blastocyst inner cell mass cell (ICM) number, 144 h post insemination, G total blastocyst trophectoderm cell number (Nanog positive), 144 h post insemination, H proportion of blastocyst ICM relative to TCN, I proportion of blastocyst trophectoderm cells relative to TCN, J relative proportion of blastocyst trophectoderm cells to ICM, K representative images of blastocyst cell numbers generated from 6 h insemination window at simulated microgravity and normal gravity control, Blue cells = inner cell mass, Red cells = trophectoderm and Blue + Pink = total cell number. Data are presented as mean ± SEM, with individual culture replicates indicated by unique symbols or individual embryos (circles). N = 203 embryos assessed for fertilisation and blastocyst development and N = 51 for cell numbers from 6 replicates for normal gravity control and N = 198 embryos assessed for fertilisation and blastocyst development and N = 24 for cell numbers from 6 replicates for simulated microgravity. Normal gravity controls denoted in white, and simulated microgravity denoted in green. Statistical significance characterized as * P < 0.05, and ** P < 0.01. Parts of this image were created in BioRender. Mcpherson, N. (2026) https://BioRender.com/tldfsf8.
Discussion
With the recent advancements in space travel and international interest in deep space exploration, Mars settlement and Moon mining, it is critical to investigate the effect of microgravity on early fertilization events not only for creating viable food sources, but also maintaining human space settlements, without the need to continually re-populate from Earth. Utilizing a purpose-built gamete and embryo culture suitable 3D clinostat we have shown that simulated microgravity impacts some functionality of sperm, resulting in reduced navigational ability, fertilization and blastocyst development/quality although severity is dependent on mammalian species and total length of microgravity exposure. Interestingly, despite the clear physiological impacts simulated microgravity has on gametes and zygotes, many embryos were still able to form healthy blastocysts, even when fertilized under these conditions.
Overall, we found similar effects on spermatozoa from humans and mice when exposed to simulated microgravity, with the greatest influence being a reduced sperm navigational ability (Figs. 1B, G, 2B). Spermatozoa have previously been shown to utilize several mechanisms to navigate through the female reproductive tract including chemotaxis (chemical signals)37, rheotaxis (fluid movement)38, and thermotaxis (change in temperature)39. Whilst human sperm was shown to be responsive to gravity independent of rheotaxis back in the 1980’s40,41, it is not considered a primary navigational force to date42. We are the first to show that gravity or ‘lack of’ also known as Gravitaxis43 is an important factor in spermatozoa ability to navigate through a channel. We theorize two ways in which gravity may contribute to sperm transit within the female reproductive tract; (i) tracking of surfaces to successfully reach the site of fertilization and (ii) regulation of inter-cellular communication. Sperm are known to accumulate near surfaces, such as the microvilli found throughout the female reproductive tract, which aid in their orientation and guidance toward the site of fertilization. This behavior is influenced by a combination of hydrodynamic forces, sperm head morphology, and their natural tendency to tilt toward surfaces44,45,46. The behavior of spermatozoa near surfaces is influenced by their orientation; maintaining a consistent orientation allows them to reach a stable state, propelling at a specific distance from the surface. However, changes in gravitational forces can disrupt this balance, causing detachment from the surface and a loss of spatial positioning41. As such, gravity can directly influence sperm orientation, likely to play a role in either attracting or repelling them from surfaces, as well as aligning their direction of movement40. Sperm possess various mechanosensors, including the LINC complex47 and several ion channels (CatSper, Slo3, P2X2, and K+ gated channels)48,49, which play crucial roles in regulating tail beating, hyperactive motility, membrane transport and polarization, intracellular pH and ion balance and the process of capacitation49. Because mechanosensors convert physical forces, such as gravity into cellular signals50, the absence of gravity is expected to disrupt these chemically regulated processes. For example, in mice, 6 h of exposure to simulated microgravity significantly reduced the mRNA content of cytoskeletal protein-encoding genes, such as Tuba1c and Tubb4b51. In our study, while minimal changes in sperm motility were observed in humans and mice at 1 and 2 h of simulated microgravity (Figs. 1C, D, 2C, D), respectively, a decline in progressive motility was evident in mice by 4 h (Fig. 2D), along with a likely reduction in pig sperm capacitation at both 2 and 6 h (Fig. 2K, L). These findings further support the idea that simulated microgravity is likely disrupting chemical signaling through sperm mechanosensors, with effects appearing to be both time-dependent and species-specific. However, the underlying mechanisms remain to be fully elucidated.
In this study, we found that we could recover human sperm navigational abilities with the introduction of a key chemoattractant progesterone (Fig. 1G). Progesterone, secreted by cumulus cells surrounding the oocyte, induces a calcium influx through the CatSper channel, initiating a signaling cascade that enhances sperm motility, and orientation towards the oocyte26. While progesterone’s chemoattractant properties work at picomolar concentrations52, we found that we could only recover sperm navigational abilities at 100 µM. The necessity for a higher concentration of progesterone in our experiments may be attributed to the limited passive diffusion of the hormone across the microfluidic channel within the 1 h experimental timeframe. Interestingly, despite the short exposure to microgravity (4–6 h) in our fertilization experiments, fertilization was able to be achieved in both mice and pigs (Fig. 3B, 5B), although decreased, suggesting that passive diffusion of progesterone from COCs might have provided an adequate local concentration to facilitate sperm navigation and fertilization under microgravity. This effect became even more apparent when microgravity exposure was extended to approximately 24 h in mice, with fertilization rates returning to levels comparable with those observed under normal gravity (Fig. 4B). However, it is also possible that the extended duration simply increased the likelihood of sperm reaching the oocyte by chance. These findings underscore the importance of chemotactic sperm responses under microgravity.
Interestingly, our results indicate that sperm may possess adaptive processes that enable them to reach the site of fertilization even in the absence of gravity. While simulated microgravity significantly reduced the number of sperm reaching the end of the microchannel, a notable subset still successfully traversed the channel regardless of the presence of progesterone or a cumulus–oocyte complex. Even more intriguing was the observation that sperm capable of reaching the end of the channel exhibited favorable characteristics, such as enhanced hyaluronan binding of human sperm, which is known to be predictive of sperm quality28,29,30,31,32 (Fig. 1H). Additionally, sperm that successfully fertilized an oocyte under simulated microgravity produced blastocysts with indicators of improved developmental potential, as evidenced by increased allocation of cells to the inner cell mass (made up of both epiblast and primitive endoderm)53 in pigs (Fig. 5F) and elevated numbers of fetal epiblast cells in mice54 (Fig. 3G). Epiblast cell numbers ( > 7 cells) are strongly correlated with reactivation potential and epiblast development is a key determinant of successful gastrulation and subsequent viability33,34,35. This suggests that the most competent sperm may possess either a greater abundance of gravity-sensitive mechanosensors, alternative types of mechanosensors that remain functional under microgravity, or the ability to switch to other environmental sensing mechanisms when gravitational cues are absent. We know that microgravity exposure in other cells can enhance quality and behaviors. For instance, microgravity has been shown to enhance stem cell regeneration, increased differentiation, proliferation, and self-renewal55,56. These findings raise the possibility that, much like stem cells, a subpopulation of sperm may not only tolerate but adapt functionally to microgravity, potentially leveraging alternative sensing mechanisms to maintain fertilization competence in extreme environments.
The timing of microgravity exposure appears to have markedly different effects on pre-implantation embryo development and quality, depending on whether it occurs during sperm transit and fertilization or during the first 24 h of zygote formation. Simulated microgravity during the 4–6 h of sperm transit and fertilization led to reduced fertilization rates in both mice and pigs, with no impact on blastocyst formation in mice, a decrease in pigs, and an increase in inner cell mass and epiblast lineage allocation in both species (Figs. 3 and 5). However, extending microgravity exposure to 24 h post-fertilization appeared more detrimental, resulting in reduced blastocyst formation, delayed early cleavage events, and a global decrease in total cell numbers in mice (Fig. 4). In previous mouse experiments, exposure to simulated microgravity during insemination had no impact on fertilization nor subsequent pregnancy rates and offspring development after 1-cell embryo transfers20. These discrepancies are likely due to differences in experimental design, including the use of a horizontal clinostat in the previously published article, which is more susceptible to fluid dynamics and orientation bias due to its single-axis rotation57 as well as the timing of sperm capacitation, which occurred prior to microgravity exposure, and the direct insemination of sperm with cumulus–oocyte complexes. Another study investigating fertilization rates in mice after 6 h of simulated microgravity using a 3D clinostat also reported no significant changes19. However, as in the previous study, sperm and cumulus-oocyte complexes were co-cultured directly, and this occurred in a large flask (12.5 cm²) containing 40 mL of culture medium, which is approximately 100 times the volume typically used in clinical assisted reproductive technologies. The rotational movement within the flask likely induced fluid mixing due to the introduction of air when capping the lid, which may have mitigated the effects of microgravity. In the same study, more pronounced effects on embryonic development emerged when embryos were cultured beyond fertilization19. Specifically, 24 h of simulated microgravity exposure resulted in a 35% reduction in implantation rates following 2-cell stage transfers, consistent with the reduced blastocyst development and lower cell numbers observed in our study for the same exposure time19. The first 24 h of embryonic development are marked by profound genetic and epigenetic changes, transitioning the zygote from a single cell to a developing diploid embryo. Key events during this period include the maternal-to-zygotic transition where maternal mRNAs and proteins are degraded and replaced by zygotic gene products and zygotic genome activation58, which in mice occurs during the late 1-cell stage59, initiating transcription from the embryonic genome. Given that microgravity is known to significantly alter gene expression related to cell growth and metabolism60, both critical for early embryo development59, it is not unexpected that exposure during this window disrupts downstream blastocyst formation.
Most spaceflight studies on pre-implantation development begin at the 2-cell stage. Embryos cultured on the SJ-10 satellite showed reduced blastocyst formation, lower total cell numbers, impaired trophoblast differentiation, and increased DNA damage, mainly attributed to radiation18. Similarly, thawed 2-cell embryos cultured on the International Space Station(ISS) exhibited poor recovery, reduced blastocyst formation, and a higher arrest rate at the 2-cell stage, with fewer total and trophectoderm cells61. Confounding factors such as launch-related stress, fluid convection, and experimental variation were attributed to the findings. While studies in pregnant mice and rats have shown that these animals can maintain pregnancies during spaceflight, although with reduced birth weight, litter size, and increased neonatal mortality62,63 and 2-cell embryos can develop into blastocysts on the ISS61, mammalian reproduction in microgravity appears possible but likely impaired. It remains unclear whether embryos fertilized and developed entirely in microgravity can produce viable blastocysts capable of implantation and sustained pregnancy. Future research must investigate prolonged microgravity exposure across all pre-implantation embryo stages under tightly controlled in vitro conditions and identify the most vulnerable developmental windows, with a focus on epigenetic and functional outcomes, including offspring health.
Together, our findings highlight that while mammalian fertilization and pre-implantation development can occur under simulated microgravity, key processes such as sperm navigation, fertilization efficiency, and embryo quality are compromised in a time- and species-dependent manner. The ability of some sperm to maintain functionality and generate blastocyst with indicators of improved developmental potential under simulated microgravity suggests the involvement of adaptive or compensatory mechanisms. However, overall developmental capacity remains reduced, particularly with prolonged exposure. These insights, combined with variable outcomes from spaceflight studies, underscore the complexity of reproductive success in microgravity and the critical need for further research across all stages of early development. Understanding the molecular and mechanical sensitivity of gametes and embryos to altered gravity is essential for ensuring the long-term reproductive sustainability of humans and livestock in space.
Materials and methods
Reagents and materials
Unless specified, all reagents and materials were acquired from Sigma-Aldrich, St. Louis, MO, USA. All media were prepared at least 4 h prior to use to allow equilibration in an incubator at 37.0 °C, 6% CO2, and 5% O2 (human and mouse) or at 38.5 °C, 5% CO2, 5% O2 (pig). Gamete and embryo handling/assessments follow our previously published methods64.
Gamete acquisition and ethics approval
Human biological material (semen) was obtained in compliance with The National Statement on Ethical Conduct in Human Research (2007, updated 2023). Semen collection was approved by the University of Adelaide Human Research Ethics Committee (H-2021-004, H-2024-006, H-2024-067), with all participants providing informed consent and reimbursed for their time. Recruited men from the public were generally healthy, aged between 18 and 50 years and who had not previously had a vasectomy/vasectomy reversal, or any condition that may affect their fertility (i.e., undescended testes, or genetic condition such as Klinefelter’s syndrome). Men were asked to abstain from ejaculation for 2–5 days prior to collection and all samples were processed within 1 h of ejaculation.
All experiments using mice followed the Australian Code for the Care and Use of Animals for Scientific Purposes (2013) and we have complied with all relevant ethical regulations for animal use. These were approved by the University of Adelaide Animal Ethics Committee (M-2023-005). Pre-pubertal (3–5-week-old) C57BL/6 x CBA (CBAF1) female mice were used for oocyte donors, and both CBAF1 and C57BL/6 male mice aged 3–6 months as sperm donors. Animals were housed in a temperature-controlled holding room at 21 °C, on a 12:12 h light/dark cycle. All mice were fed standard chow and acidified water ad libitum.
Sow ovaries were collected from a local abattoir and transported to the Adelaide Health and Medical Science Building (University of Adelaide) in warm (30-35 °C) saline 0.9% NaCl solution (Baxter International, Deerfield, Illinois, USA). Extended pig semen was purchased from local boar studs (Sabor Artificial Breeding Center, Clare, SA, Australia).
Preparation of spermatozoa and baseline motility analysis
Human sperm was isolated from whole semen through gentle washing via centrifugation at 400 x g for 5 min, to remove the viscous seminal plasma and the sperm pellet resuspended in G-IVF PLUS (Vitrolife, Gothenburg, Sweden).
Mice were humanely killed by cervical dislocation, and sperm extracted from the cauda epididymis and vas deferens in G-IVF PLUS medium.
Pig semen was centrifuged at 400 x g for 5 min to remove the extender, and the sperm resuspended in supplemented M199 sperm wash medium (Sigma-Aldrich, St. Louis, Missouri, USA) before centrifugation at 400 x g for 5 min. The supernatant was discarded, and the sperm pellet resuspended in M199 medium before further diluting to a concentration of 5 × 106 sperm/mL in TALPS fertilization medium (Sigma-Aldrich).
Baseline sperm motility and concentration were assessed using the Microptic Computer-Assisted Semen Analyzer (CASA; Microptic, Barcelona, Spain), with at least 500 spermatozoa assessed across five or more fields of view.
Microgravity simulation with random positioning 3-D clinostat
A dual axis 3-D Clinostat (Firefly Biotech, Adelaide, SA, Australia)65 within a standard dual-gas incubator (Heracell VIOS 160i; Thermo Scientific, Waltham, Massachusetts, USA) was used to stimulate microgravity for three-dimensional gamete and embryo culture (see Supplementary Fig. 1A-B and Supplementary Video 1). Briefly, two axes driven by two independently operated control engines constantly reorientate the sample chamber to random positions to generate gravity nullification (below 0.1 g within 2 min). The random walk algorithm utilized was as previously described24 and the full algorithm presented in Supplementary Data 1. The rotational control algorithm utilized was as previously described66, and the full algorithm presented in Supplementary Data 2. Gravity bias is a proprietary system developed by Firefly Biotech and we acknowledge that the reliance on a proprietary device may present a limitation in reproducibility. The system sample chamber supports the specialized micro-channel µ-Slide VI 0.4 (Ibidi GmbH, Grafelfing, Germany).
Sperm microgravity swimming challenge
The µ-Slide VI 0.4 (Ibidi GmbH) was prepared with 100 µL of G-IVF PLUS medium. To avoid air bubbles and ensured a closed loop system, the channel entry was blocked using a male leur plug (Sigma-Aldrich) with 50 µL of diluted sperm solution (human, mouse, or pig), while the opposite end was blocked with 50 µL of G-IVF PLUS (human and mouse) or TALPS fertilization media (pig) only. Spermatozoa were incubated for 1 h (human) 2 and 4 h (mouse) and 2 and 6 h (pig), in either a rack of the dual gas incubator (normal gravity) or the 3-D clinostat (microgravity). At the end of the incubation period, the end plug was removed to assess if sperm could swim though the channel to the opposite end. An aliquot of solution was taken for sperm recovery and motility analysis using Microptic CASA. For mouse and pig time-course experiments, sperm samples were collected at 1, 2, and 4 h (mouse) and 2 h and 6 h (pig).
For chemoattraction experiments, either 10 µM or 100 µM of progesterone dissolved in G-IVF-PLUS (P8783, batches of 100 mM dissolved in Ethyl alcohol, Pure (200 proof, anhydrous, ≥99.5%, 459836, Merck, New Jersey, USA)) were placed in the end plug.
Sperm binding capabilities were assessed using the HBA® hyaluronan binding assay (CooperSurgical Fertility Solutions, Knardrupvej, Denmark). A 10 µl aliquot of sperm recovery was incubated for 15 min at 37 °C. A total of 200 sperm were counted and classified as either bound (head attached and tails moving) or unbound (freely moving), and the proportion of sperm bound to hyaluronan calculated.
Mouse oocyte collection, in vitro fertilization microgravity challenge and embryo culture
Female mice were super-ovulated by intra-peritoneal injection of 5IU of PMSG (Folligon; Merk, Darmstadt, Germany), followed by injection of 5IU of HCG (Pregnyl; Merk) 46–48 h later. At 15 h post-hCG injection, cumulus-enclosed oocytes were collected by puncturing the oviduct and two COC bunches ( ~ 30 oocytes) were added to 100 µL of pre-equilibrated G-IVF PLUS (Vitrolife) within the µ-Slide VI 0.4 (Ibidi GmbH), prior to the addition of 10,000 sperm (50 µL in male leur plug).
Gametes were co-incubated for 4 h, either as normal gravity controls or as a microgravity experiment, where both spermatozoa and COC were exposed to microgravity conditions.
Putative zygotes were then grouped cultured (n = 10 per drop) under paraffin oil in 20 µl pre-equilibrated G-TL culture medium (Vitrolife) for 24 h to assess fertilization by cleavage to the 2-cell stage (day 2). Unfertilized oocytes or oocytes with evidence of fragmentation were separated from drops with 2-cell embryos. Embryo were either cultured for 96 h to assess blastocyst formation and expansion (day 5) or placed in an Embryoslide culture dish (Vitrolife) and incubated in an Embryoscope time-lapse system (Vitrolife) to analyze developmental kinetics. Blastocysts were categorized as either early blastocyst (blastocoel cavity <2/3 of embryo), blastocyst, expanded blastocyst (blastocoel cavity >2/3 of embryo) or hatching blastocyst (protrusion of the blastocyst through the zona pellucida) while morphokinetic markers of embryo development were assessed using the inbuilt Vitrolife system.
For some experiments, gamete co-incubation during IVF was extended for 24 h. Fertilization (2-cell cleave) was assessed upon removal from the µ-Slide, with 2-cell embryos being placed together to continue standard culture until day 5 or in the Embryoscope time-lapse incubator for embryo morphokinetic assessment as above.
Mouse blastocyst cellular allocation by immunofluorescence
Blastocyst cell differentiation (inner cell mass and epiblast cells) was assessed at 96 h of culture67,68,69,70. Day 5 blastocysts were fixed overnight in 10% formalin in Phosphate Buffer Saline (PBS) at 4 °C. Fixed blastocysts were incubated in 0.1 M glycine for 5 min before being permeabilized in 0.25% Triton X-100 for 15 min at room temperature (RT), then blocked overnight with 10% donkey serum at 4 °C. Blastocysts were then incubated in rabbit anti-Nanog (Cosmo Bio, Japan, Cat# REC-RCAB0002P-F, RRID:AB_10706358) (1:200) and goat anti-Oct4 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-8628, RRID:AB_653551) (1:100) for 1.5 h at 37 °C. The negative control was incubated in 1:200 rabbit serum and 1:100 goat serum instead of the primary antibodies. Blastocysts were washed with PBS, then incubated in donkey anti-rabbit Alexa 488 (Life Technologies, Carlsbad, CA, USA, Cat# A-21206, RRID: AB_2535792) (1:100) and donkey anti-goat Alexa 594 (Life Technologies, Cat# A11058, RRID:AB_2534105) (1:100) for 2 h at RT. Lastly, blastocysts were incubated in DAPI for 23 min at RT before being loaded in glycerol and counted using a fluorescence microscope (Olympus BX51 Epifluorescence Microscope, Shinjuku City, Tokyo, Japan). Total cell number was assessed by nuclei stained DAPI; inner cell mass cells stained positive for Oct4, and epiblast cells stained positive for Nanog within Oct4 positive populations.
Pig oocyte collection and in vitro maturation
Cumulus-oocyte-complexes (COCs) were aspirated from clear follicles with diameters of 3–6 mm using an 18-gauge needle. COCs that had multiple compact cumulus cell layers, and no morphological defects underwent in vitro maturation (IVM) for 39-42 h in BOMED medium (basic media 199 HEPES, 5 mg/ml insulin, 5 mg/ml FSH, 10 mg/ml epidermal growth factor, sow follicular fluid filter sterilized) (Sigma-Aldrich) at 38.5 °C, 5% CO2 and 5% O2. At the end of IVM, oocytes were denuded with hyaluronidase.
Pig in vitro fertilization microgravity challenge and embryo culture
Following hyaluronidase treatment, pig oocytes were placed into embryo handling drops (H-NCSU23, Sigma-Aldrich). Oocytes (n = 15–30) were then washed and incubated in 100 µL of TALP medium, within the µ-Slide VI 0.4 (Ibidi GmbH), prior to the addition of 5 ×105 pre-prepared sperm (50 µL placed in the male leur plug).
Following gamete co-incubation, putative zygotes were removed and denuded in embryo handling drops (H-NCSU23). Zygotes were washed using NCSU23-plg (0.2 mM pyruvate, 5.7 mM lactate, 0.6 mM glucose, non-essential amino acids, Sigma-Aldrich) before being cultured for 3 days at 38.5 °C, 6% CO2 and 5% O2. Fertilization was assessed at 56 ± 1.5 h post-insemination and was identified by cleavage to the ≥2-cell stage with minimal fragmentation. For days 4–6 of culture (96-144 h post-insemination), embryos were placed in NCSU23-G medium (5.6 mM glucose, essential and non-essential amino acids, Sigma-Aldrich). For the final 24 h of culture 10% fetal bovine serum was added to aid developmental progression. Blastocyst formation and expansion was assessed on day 6 (144 h post-insemination). Blastocysts were categorized as either an early blastocyst, blastocyst, expanded blastocyst, or hatching blastocyst.
Pig blastocyst cellular allocation by immunofluorescence
Blastocyst underwent differential staining 144 h post insemination (day 6)71. Zona pellucidae were removed with 0.5% Pronase solution (Sigma-Aldrich), and blastocysts incubated in 10-mM Picrylsulfonic acid (Sigma-Aldrich) on ice in the dark for 20 min. Blastocysts were then washed in 0.2 mg/mL anti-DNP BSA for 10 min at 37 °C, then incubated in guinea pig complement serum (1:10, Sigma-Aldrich) with 0.1 mM bisbenzimide and 20 µg/mL propidium iodide (Sigma-Aldrich) for 10 min at 37 °C. Blastocysts were fixed in 99% ethanol for 2 min, placed on a glass slide, covered with glycerol, and a cover slip. Blastocysts were viewed under an ultraviolet epifluorescence microscope (Nikon Eclipse TS100, Tokyo, Japan) with trophectoderm cells fluorescing as red and inner cell mass cells fluorescing as blue. The proportions of trophectoderm cells, inner cell mass cells and total cell numbers were then calculated.
Statistics
All measures are reported as mean ± SEM. Statistical significance was determined as indicated, by using either paired or unpaired Student’s T test, One-way ANOVA with Tukey’s multiple comparisons test or General Linear Model controlling for replicate as appropriate. Statistics software used was either GraphPad Prism v010 (GraphPad Software, La Jolla, CA, USA), or SPSS v29 (SPSS Inc., Chicago, IL, USA). A P-value of less than 0.05 was considered statistically significant, while P < 0.1 indicated a trend.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
The authors declare that all data supporting the findings of this study are available within the paper and Supplementary Data 3.
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Acknowledgements
We would like to thank Giles Kirby CEO and founder of Firefly Biotech and Ian Johnson—who provided invaluable expertise on the 3D clinostat and in vitro culture of mammalian cells under simulated microgravity. Parts of all our figures were created with BioRender.com (Academic License).
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H.E.L. and N.O.M. conceived the study. H.E.L., M.B.N., and N.O.M. designed the experiments. H.E.L., V.N., B.M.A., S.M.M., and N.O.M. performed the experiments. H.E.L. analysed the data. H.E.L., M.G., and N.O.M. interpreted the data. H.E.L. and N.O.M. wrote the original draft. All authors contributed to review and editing and approved the final manuscript.
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Lyons, H.E., Nikitaras, V., Arman, B.M. et al. Simulated microgravity alters sperm navigation, fertilization and embryo development in mammals. Commun Biol 9, 401 (2026). https://doi.org/10.1038/s42003-026-09734-4
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DOI: https://doi.org/10.1038/s42003-026-09734-4
