P-187 Profiling secreted proteins in human embryos: Time-specific discrimination of ploidy and confirmation of enhanced post-implantation development with maternal co-culture
Authors: Palomar A , Quiñonero A , Yagüe-Serrano R , Morales-Alvarez S , Molè M , Florensa M , Mercader A , Domínguez F , Sánchez-Ribas I 
Published: July 2024

Abstract

Study question
Can proteins released by embryos into culture medium between 7- and 14-days post-fertilization (d.p.f.) potentially function as biomarkers indicating the ploidy status of the embryos?

Summary answer
Combined secreted levels of calreticulin (CALR), human Placental Lactogen (hPL), Placental Growth Factor (PlGF) and human Pappalysin-1 (PAPP-A) differentiate euploid from chromosome 21-aneuploid embryos.

What is known already
Exploring post-implantation embryo development is vital due to potential errors impacting early pregnancy or postnatal health during this period. Trisomy 21 (T21) is a common aneuploidy that can lead to live births but with postnatal health concerns, while chromosome 21 monosomy (M21) often results in embryo developmental arrest. Understanding post-implantation development and secreted products into human post-implantation niche is crucial for unraveling embryo-maternal interactions. Building on prior studies indicating ploidy-related gene and metabolite expression in pre-implantation embryos (DOI:10.1016/j.fertnstert.2019.01.023), this study aims to assess variations in the secretome of embryos with different ploidy during post-implantation development.

Study design, size, duration
Comparative study involving 48 science-donated human 6 d.p.f. blastocysts initiating hatching coming from in-vitro fertilization cycles. Preimplantation genetic testing identified Euploid (n = 20), pure T21 (n = 14), and pure M21 (n = 14) embryos. Two extended in-vitro culture protocols were compared: co-culturing of human embryos in a 3D system with endometrial cells or in a culture medium designed for post-implantation development but lacking maternal cells.

Participants/materials, setting, methods
Euploid, T21 and M21 blastocysts were thawed and cultured in-vitro until 14 d.p.f. in both culture systems described above. Levels of CALR, hPL, PlGF and PAPP-A in conditioned embryo media were quantified by ELISA immunoassays at 7, 10, 12 and 14 d.p.f. Absolute secreted protein concentrations (ng/mL) in each case were compared by two-way ANOVA followed by Tukey’s test for multiple comparisons. P &lt; 0.05 was consider as statistically significant.

Main results and the role of chance
In both culture systems tested, at 7 d.p.f. secreted CALR, hPL and PlGF by M21 embryos were diminished when compared to euploid embryos (p &lt; 0.01). At 10 d.p.f., euploid embryos displayed a significant increase in secreted PAPP-A, surpassing levels in T21 (p &lt; 0.05) and M21 embryos (p &lt; 0.01). Embryo co-culture in 3D endometrial systems led to progressive increase in secreted PAPP-A and PlGF by euploid embryos at 14 d.p.f. compared to earlier timepoints (7 d.p.f.,p&lt;0.01; 10d.p.f.,p&lt;0.05). PAPP-A secretion did not increase in T21 embryos during extended culture. T21 embryos demonstrated decreased PAPP-A secretion at 10, 12, and 14 d.p.f. compared to euploid embryos (p &lt; 0.01 in all cases). Secreted PlGF in T21 embryos rose significantly at 12 (p &lt; 0.05) and 14 d.p.f. (p &lt; 0.05), despite consistently lower levels than euploid embryos (p &lt; 0.01). All studied proteins showed significantly diminished secretions during post-implantation development in M21 embryos compared to euploid ones. Comparing culture systems, the absence of maternal cells led to a significant decrease in secreted protein levels and viability impairment beyond 11 d.p.f. Consequently, only two timepoints (7- 10d.p.f.) were considered for analysis in this culture system. The findings underscore the potential of these secreted biomarkers for distinguishing embryo ploidy, with maternal co-culture supporting enhanced developmental outcomes.

Limitations, reasons for caution
This is an in-vitro pilot study that should be validated in larger cohorts.

Wider implications of the findings
Secretions of CALR, hPL, and PlGF at 7 d.p.f. effectively distinguish M21 embryos, while PAPP-A and PlGF secretions at 10-12 d.p.f. are indicative of embryo ploidy. Additionally, co-culturing with maternal cells notably promotes proper embryo development, particularly enhancing trophoblast differentiation and secretion of trophoblast-related proteins. Funding: ISCII(PI20/00405;PI23/00860;FI18/00009;MV21/00086); GVA (CIAICO/2022/203;CIAPOT/2022/18) and IDI-20210916

Trial registration number
Not Applicable