P-328 Uterine Fluid microbiome composition in fertile controls and infertile women
Authors: Carnemolla G , Russo E , Giacomini E , Pavone V , Ferraro C , Di Stefano G , Biancolini D , Vanni V  S , Barberis N , Sterpi V , Benaglia L , Marzanati D , Rovere-Querini P , Vigano’ P , Riba M 
Published: July 2024

Abstract

Study question
Is the microbiome of the Uterine Fluid (UF) related to outcomes of frozen-thawed blastocyst transfer?

Summary answer
Microbiome of UF collected by double-sheated catheter shows Lactobacilli as the most abundant species but does not relate to outcomes of frozen-thawed blastocyst transfer.

What is known already
The microbiome of UF has been described to be largely dominated (&gt;90%) by Lactobacilli and has been shown to be markedly associated with the outcomes of embryo transfer. Nonetheless, previous reports have not collected UF by avoiding vaginal/cervical contamination through a double-sheated catheter.

Study design, size, duration
Observational study conducted at Fondazione IRCCS Ca’Granda Ospedale Maggiore Policlinico and IRCCS Ospedale San Raffaele, Milan, Italy. n = 13 fertile controls underwent UF sampling in the pre-receptive (LH + 2) and receptive (LH + 7) phase of a monitored natural cycle and n = 48 infertile women underwent UF sampling on day LH + 7 of the monitored natural cycle preceding that of their first frozen-thawed blastocyst transfer. UF was sampled by aspiration with a double-sheated catheter (commonly used for embryo transfer.

Participants/materials, setting, methods
Microbiome analysis was conducted following the Illumina protocol for V3-V5 regions of the 16S rRNA and using the Illumina MiSeq instrument. Raw sequencing data processing encompassed quality controls, and QIIME 2 scripts were employed to obtain taxonomic abundances (taxonomic levels L2-L7). Alpha (within sample), and beta (among samples) diversity and differential expression at taxonomic level (using limma, Bioconductor library) were calculated.

Main results and the role of chance
UF microbiome analysis showed lack of separation between samples from the two Centres, confirming the reproducibility of the technique. UF microbiome was also found to be stable throughout the menstrual cycle, with no diversity observed between LH + 2 and LH + 7 samples in the population of fertile controls (p = 0.9, Adonis test on beta diversity; confirmed by differential abundance tests based on linear regression models at taxonomic levels). UF of fertile controls showed Lactobacillus Helveticus as the most abundant species (16.2%), followed by other Lactobacilli (unspecified) (12.1 %), Lactobacillus Iners (11.8%), Streptococcus (unspecified) (6.8%), Bifidobacterium (4.7%) and Gardnerella (1.9 %). In women who achieved successful implantation after FET, the most abundant species was L. Helveticus (58.1%) followed by L. Iners (9.8%), Acinetobacter lwoffii (5.9%) and other Lactobacilli (unspecified) (4.0%). No statistically significant differences were found in linear regression comparisons at taxonomic level with UF of women with failed implantation, in whom most abundant species were L. Helveticus (50.4%, p = 0.11), L. Iners (15.8%, p = 0.95), other Lactobacilli (unspecified) (13.7%, p = 0.17) and Garnderella (6.5%, p = 0.19). The proportion of Lactobacillus- dominated (&gt;90%) samples was 50% (n = 10/20) in infertile women achieving successful implantation and 46% (n = 13/28) in infertile women with failed implantation (p = 0.8).

Limitations, reasons for caution
These results are preliminary and only include the first n = 48 patients of the total n = 249 patients that will be enrolled. A larger sample is required to further investigate possible relations between UF microbiome composition and implantation outcomes.

Wider implications of the findings
As UF microbiome was found to be stable between cycle phases and it is non-invasively collected, it could be analysed in the same cycle in which ET is scheduled. This might increase accuracy compared to current tests for endometrial receptivity, that have to be performed in the cycle before ET.

Trial registration number
RF-2019-12369460