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

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.

The embryo culture media in the era of epigenetics: is it time to go back to nature?

“…In this species, we also created a biobank of reproductive fluids and have a small number of animals in a sanctuary to study their long-term health and the effects of assisted reproductive techniques. Finally, our group completed a preliminary human clinical trial in which the embryo recipient mothers themselves donated uterine fluid to be added to the medium for culturing their own embryos (Canha-Gouveia et al, 2021). As a result, the first two children from embryos developed in culture media containing their mother's own uterine fluid were born and we also established all the necessary regulatory and biosafety conditions to create a biobank of human reproductive fluids that can be used for future research (Figure 4).…”

“…In all three species, the fluids were collected ex vivo (slaughterhouse specimens in pigs and cows, surgically removed organs in women) or in vivo (transvaginal aspiration of endometrial fluid). The fluid volumes obtained were usually between 20 and 100 microlitres, depending on the organ and the species, and the samples were immediately transported to the laboratory, where purification by centrifugation and quality control tests were performed (Canha-Gouveia et al, 2021) before storing the different batches at the corresponding biobank (human or animal). In general, fluids are added to the culture media at ~1-2% v/v and all the mentioned pieces of evidence have been obtained under these conditions.…”

Programming of Embryonic Development

“…Coy and colleagues have shown that oviductal fluid, as well as specific proteins secreted by the oviduct, improve the rates of fertilization and embryonic development in vitro and in vivo, resulting in greater pregnancy rates and improved pregnancy outcomes with ART [110][111][112][113]. This technology holds great potential to overcome poor fertility and also to maximize the impact of elite genetic stock, and thus to dramatically improve the efficiency of livestock production.…”