Physicochemical and Functional Characterization of Female Reproductive Fluids: A Report of the First Two Infants Born Following Addition of Their Mother's Fluids to the Embryo Culture Media.

Analuce Canha-Gouveia,Pilar Coy,Juan Carlos Martínez-Soto,Maria Teresa Prieto-Sánchez,Evelyne París-Oller,Cristina Soriano-Úbeda,Maria Luisa Sánchez-Ferrer,José Landeras,Marta Mollá

doi:10.3389/fphys.2021.710887

Abstract

Culture media supplemented with reproductive fluids (RF) have been used in livestock species, improving the efficiency and quality of in vitro produced embryos. However, usefulness in humans is still unknown. In this study, we collected human reproductive fluids (HRFs) ex vivo (from 25 patients undergoing abdominal hysterectomy plus bilateral salpingectomy) and in vivo (from 31 oocyte donors). Afterward, protocols to evaluate their osmolality, pH, total protein concentration, endotoxin level, and sterility were optimized, establishing security ranges for their use as natural additives. In addition, a functional assay was developed with bovine embryos grown in vitro in a medium supplemented with 1% of collected HRFs. Finally, a proof of concept was performed with six patients on post ovulation day 2 to evaluate the full-term viability of embryos grown in media supplemented with autologous uterine fluid, collected under in vivo conditions. Two of the embryos resulted in successful pregnancy and delivery of healthy babies. In conclusion, this study establishes a complete quality control sheet of HRFs as additives for embryo culture media and shows first preliminary data on obtaining healthy offspring derived from embryos grown in media supplemented with HRFs.

Highlights

Female reproductive fluids (RFs) are physiological “culture media” where first events in the life cycle take place: oocytes grow and mature inside the follicular fluid; a mature oocyte is fertilized by sperm in the oviduct, where gametes are surrounded by the oviductal fluid (Avilés et al, 2010)
The quality control (QC) sheet for human reproductive fluids (HRFs) collected in vivo and ex vivo to be used as new natural products in culture media was achieved by the characterization of non-diluted human oviductal fluids (HOFs) and human uterine fluids (HUFs) in patients and donors
The ex vivo collection of HUFs and HOFs was successfully performed in 25 patients with an average age of 45 years, and in vivo collection (HUFs) was successfully performed in 31 oocyte donors with an average age of 25 years (Table 1)

Summary

Introduction

Female reproductive fluids (RFs) are physiological “culture media” where first events in the life cycle take place: oocytes grow and mature inside the follicular fluid; a mature oocyte is fertilized by sperm in the oviduct, where gametes are surrounded by the oviductal fluid (Avilés et al, 2010). After fertilization and first cell divisions, the embryo moves toward the uterus around days 3–5 post-fertilization, where endometrial glands secrete uterine fluid, which, is essential for embryo implantation and survival (Zhang et al, 2017) These RFs have been widely studied to Biofluids as Culture Media Supplements identify novel markers of endometrial receptivity and gain significant insights into the mechanisms underlying unexplained infertility, recurrent pregnancy losses, and other endometrial pathologies (Salamonsen et al, 2013; Bhusane et al, 2016). It is crucial to establish security ranges for all these parameters in HRFs, especially human oviductal fluids (HOFs) and human uterine fluids (HUFs) in which gametes and embryos develop naturally

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