Gut microbes are important participants in human metabolism, and their biochemical activities and metabolites are inextricably linked to human health. As the largest and most complex microecosystem of the human body, the gut microbiome itself and its metabolites can not only regulate human health, but also play an important role as a bridge between diet and host. As Nobel laureate Joshua Lederberg has pointed out, the human body and its symbiotic microorganisms constitute superorganism.
With the exploration of the role of gut microbes, there is continuous evidence that microbiome imbalances trigger physiological responses in somatic tissues. However, little is previously known about the microbiome's perturbation of the reproductive system.
2024年5月1日,欧洲分子生物学实验室(EMBL)的Jamie A.Hackett等人在《Nature》上发表一篇名为《Paternal microbiome perturbations impact offspring fitness》的研究论文,表明肠道微生物群是小鼠父系孕前环境和代际健康之间的关键界面。
Perturbation of the gut microbiota of the intended father increases the likelihood of low birth weight, severe growth restriction, and premature death in his offspring. This effect is associated with a dynamic response to dysbiosis induced in the male reproductive system, including impaired leptin signaling, altered testicular metabolite profiles, and repositioning of small RNA payloads in spermatozoa. An adjustable "gut-germline axis" was defined in the study, which is very sensitive to environmental exposures and alters the health of offspring by influencing placental function. (Figure 1)
The researchers used non-absorbable antibiotics (nABX) to establish an induction model of gut microbiota dysbiosis in isogenic male mice. 16S ribosomal RNA sequencing showed that 6 weeks of treatment with low-dose nABX resulted in a reversible decrease in gut microbiota diversity, abundance and richness, but this dysbiosis had no significant effect on male body weight, fertility or survival, and could gradually recover after 8 weeks of nABX discontinuation. (Fig. 2)
1. The impact of paternal dysbiosis on offspring
Neonates (F1) of nABX fathers had significantly lower birth weight than offspring of nABX fathers, and their mean weight was also reduced throughout development. In addition, a predominant, but only partially expansive, postnatal phenotype was observed in F1, manifesting as severe growth inhibition (SGR), which was absent in the offspring of the control group. (Fig. 3)
and, the offspring of F1 treated with nABX had a significantly increased postnatal mortality compared to the offspring of the control group. This occurs predominantly in SGR offspring, suggesting that increased mortality is associated with increased susceptibility to F1 for growth restriction.
Transcriptome analysis of SGR progeny born to nABX males suggests that differentially expressed genes (DEGs) in the brain and brown adipose tissue (BAT) are preferentially enriched in the reactive pathway involved in metabolic processes. These results indicate that there are intergenerational effects of paternal dysbiosis on offspring growth, metabolic network and survival, and these phenotypes are probabilistic rather than deterministic responses, and therefore manifest as changes in offspring health risks.
Next, the researchers explored several parallel strategies to disrupt the parent microbiota, and the results were similar to those of nABX. In this regard, a number of different perturbations in the gut microbiota of the intended father increase the risk of developmental disability and premature death in the offspring, which supports a direct link between paternal dysbiosis and the health of the offspring. (Fig. 4)
2. Reversibility of paternal effects
The researchers explored whether paternal recovery from intestinal dysregulation could save the F1 phenotype. The convalescent father after nABX discontinuation still had an effect on F1 offspring, and when the microbiome in vivo recovered, the weight phenotype of F1 neonates recovered and grew normally.
Transcriptomics showed that SGR offspring born to nABX fathers exhibited highly similar ontological enrichment of genes to independent offspring born to convalescent fathers, suggesting a common underlying etiology. In addition, further detection of SGR progeny showed no F2 effect of transmission. These data suggest that the F1 phenotype, caused by dysbiotic fathers, is not due to genetic inheritance and is not transmitted after the first generation. (Fig. 5)
3. The way of intergenerational transmission
To confirm how it is inherited from generation to generation, the researchers first considered whether there was paternal transmission in the gut microbiome itself. Studies have shown that the altered paternal microbiota is not transmitted to the mother and offspring, and that the post-representative type is associated with the paternal microbiota but not the microbiota itself.
A series of experiments allowed the researchers to determine that neither altered parental microbiota nor indirect responses in the parent were the basis for the F1 effect. As a result, researchers began to perform in vitro fertilization (IVF) to see if the F1 phenotype was specifically transmitted through paternal gametes. Subsequently, it was found that the paternal-induced F1 phenotype appeared in an independent uterine genetic background and was mainly transmitted through gametes and coplasmic molecules.
4. Gut-germline axis
The phenomenon of transmission through germline has led researchers to focus on the physiological changes in the paternal reproductive system induced by acute gut microbiota dysbiosis. Testicular mass was observed in nABX males that was significantly smaller than in the control group, with structural changes, suggesting that the testes were physiologically affected by intestinal microbiota dysbiosis.
At the molecular level, significant separation was observed between the nABX phase and the convalescent phase and the control testes, while there was no difference in the testicular metabolome profiles with complete microbial recovery, suggesting that the dynamic recovery of metabolites occurred simultaneously with the restoration of the gut microbiota and the reversal of the F1 passage effect.
The researchers also found 68 significantly differentially abundant metabolites in the testes of dysbiosis male animals, all of which are involved in germ cell function. The authors further investigated the transcriptome profile of male testes with dysbiosis and observed limited expression changes at the gross and unicellular levels. Genomic enrichment showed that genes related to glycerophospholipid and steroid production were preferentially dysregulated, consistent with alterations in metabolomic signatures. The most sensitive gene is leptin, which encodes a hormone that is produced primarily by fat cells, but is also produced by germ cells, which plays a key role in energy balance and reproduction.
Cumulative evidence suggests that disturbances in the gut microbiota lead to significant changes in the testicular environment, including alterations in metabolite profile, physiology, and hormones. This suggests that there is an entero-reproductive axis in mammals that has an important homeostatic function. One of the paternal responses to nABX is a strong dysregulation of leptin. The data suggest that leptin is systematically regulated by inducible microbiome dysregulation, and that direct perturbation of paternal leptin before conception will have an intergenerational impact on the gene expression program of the offspring, suggesting that leptin is an important signaling component of the gut-germ axis. (Fig. 6)
Next, the authors sought to understand the effect of the gut-germline axis on mature gametes by looking for molecular changes in spermatozoa. Overall, while DNA methylation is relatively stable, the small RNA composition in spermatozoa is altered in nABX-mediated dysbiosis. Taking into account the alterations in metabolites and hormone profiles, this suggests that complex changes in macromolecular composition are passed on to future generations.
5. Patrilineal dysbiosis affects the placenta
Starting from the initial source of embryonic defects, the researchers explored the mechanism by which sperm influenced post-representation.
The embryos did not have DEGs compared to the control group, and in contrast, the placental transcriptome exhibited strong aggregation, dependent on the paternal nABX mechanism. Gene expression analysis highlighted alterations in gene expression associated with glycolytic metabolic processes, prolactin and steroid molecule metabolism, and several regulators of placental development, such as genes Hand1 and Syna, which are associated with impaired placental ontogeny.
To further investigate the possibility of placental insufficiency induced by fathers with dysbiosis, the researchers examined placental structure. A significant reduction in the placental labyrinth area, marked impairment of vascularization, and an increase in placental infarction from nABX fathers were found. The level of placental growth factor (PLGF) hormone is the main diagnostic indicator of preeclampsia in humans, and the PLGF in the placenta and the sFLT/PLGF ratio are significantly increased in the placenta in the adverse male offspring induced by microbiota. (Fig. 7)
Taken together, there is cumulative evidence that environmentally-induced disturbances in the gut microbiota of intended paternal fathers lead to associated reproductive effects. This suggests that there is a regulatory gut-germline axis that, when disturbed, can be transmitted to affect the risk of disease in offspring, partially mechanistically affecting the upcoming placental function.
Thus, the gut microbiota can serve as a major interface where different environmental inputs, such as antibiotic regimes or diet, can converge directly or indirectly and signal to male germ cells, ultimately influencing the offspring.
In addition, restoring the paternal gut microbiota prior to conception can rescue the F1 phenotype, suggesting that the effects of gut microbiota on reproduction are remedy. Given the prevalence of lifestyle and antibiotic practices, this may prove to be an area of interest in mitigating adverse pregnancy outcomes.
The researchers mentioned that the experiment answered the question of how environmental factors alter complex biological systems at scale, from direct molecular responses to intergenerational disease susceptibility, and emphasized the importance of understanding this question.
DOI：https://doi.org/10.1038/s41586-024-07336-w