P-159 Impact of the symbiotic metabolites D-amino acids on preimplantation development
Authors: Fukuoka M , Yamada M , Sasabe J , Miyado K , Tanaka M 
Published: June 2023

Abstract

Study question
Are the metabolites derived from the symbiotic microbiota of reproductive tract involved in preimplantation development?

Summary answer
The metabolites derived from the symbiotic microbiota of reproductive tract, D-amino acid（D-AA）, contributed to preimplantation development.

What is known already
It has been known that Lactobacillus exist in the vagina as symbiotic bacteria. Though it was thought that Lactobacillus keep the pH environment acidic and prevent infection by pathogenic bacteria, thereby keeping the uterus and fallopian tubes sterile in mammal, we have identified Staphylococcus aureus in oviductal lavage fluid from female mice and confirmed that the antibiotics disrupt the implantation pattern.
In human, the vaginal microbiota is reported to be dominated by Lactobacillus, which produces D-amino acids (D-AA). These findings suggest the involvement of bacterial metabolites D-AA in the establishment of human pregnancy.

Study design, size, duration
23 women undergoing in vitro fertilization (IVF) treatment at Keio University Hospital from 2019 to 2021 were assessed for eligibility. 3 were excluded due to spontaneous pregnancy or infectious disease. Twenty subjects were allocated to the successful pregnancy group (S-group (8); mean ± standard deviation 35.5 ± 2.8 years) defined as gestational sac detected on ultrasound at 5 weeks' gestation; and to the unsuccessful pregnancy group (U- group (12); 35.5 ± 2.7 years)).

Participants/materials, setting, methods
Genomic DNA (gDNA) extracted from vaginal and intrauterine samples at each menstrual cycle was used for metagenome analysis at the genus and the species levels by next-generation sequencing, and by quantitative real-time PCR. Amino acids were analyzed using a two-dimensional HPLC.
Immunocytochemical staining by a polyclonal antibody to D-AA oxidase (DAO), which catabolizes D-AA, was performed using B6D2F1 mice and mice with a natural point-mutation G181R in DAO gene, which abolishes DAO activity.

Main results and the role of chance
Both vaginal/uterine bacterial counts were higher in the luteal phase than in the follicular phase. Vaginal bacterial counts in the S-group were lower than those in the U- group. The composition of microbiota of the vaginal samples (N = 23) shifted from the follicular to the luteal phase, with Lactobacillus spp. predominating. However, there was no significant difference in the ratio of Lactobacillus spp. between the S- and U-groups. The intrauterine samples of the S-group contained a high abundance of L. crispatus, L. gasseri, and L. ultunensis, while the U-group contained a high abundance of L. jensenii.
Next, we analyzed the Lactobacillus metabolite, D-AA, and detected D-Ser, D-Ala, D-Asn, and D-Pro in the vaginal, uterine, and follicular fluid of the S-group. Follicular fluid that developed into blastocysts contained more D-AA than that of arrested embryos (N = 2). To investigate the significance of D-AA in the preimplantation development, mouse embryos were cultured in amino acid-depleted medium. All of them arrested (0.0%: 0/78), whereas 41% (25/61) of the embryos with D-AA added to the amino acid-depleted medium developed into blastocysts. We confirmed the expression of DAO in oocytes and blastocysts. DAO-null mice had fewer births than wild type.

Limitations, reasons for caution
The main limitation is the differences of symbiotic microbiota in reproductive tract and embryonic development between mice and humans; it may be difficult to apply the results of this analysis to humans.

Wider implications of the findings
Our results display a correlation between species-level microbiome profiling and IVF outcomes. In addition, microbiota-derived metabolites D-AA are suggested to be crucial mediators of embryo development. These findings could give rise to the development of new diagnostic techniques and drug.

Trial registration number
Not applicable