Cervicovaginal fluid (variar sigla MCU)

The MCU is a mixture of oviductal, uterine and cervical fluid that has protective functions, through the innate immunity provided by mucins, also serving as a mechanical barrier and facilitating the transport of sperm into the cavity. uterine [2]. This fluid is easily accessible in a non-invasive way and changes in its characteristics are currently used to detect, with great accuracy, the fertility window in natural methods for its regulation [1].

 

After ovulation and especially immediately after fertilization, the oviductal, uterine and MCU fluid increase several times their production and flow downstream [3]. From the moment of fertilization, a rich molecular dialogue begins between the mother and the embryo that takes place in the oviduct. This molecular dialogue is extremely synchronized and has the purpose of favoring the development of the embryo in its various stages, in addition to facilitating transport to the implantation site in the uterus [4].

Later and during the implantation process, this molecular exchange is transferred to the endometrium, in the uterus, beginning around the 6th day after fertilization. In other words, part or all of the molecules secreted by the embryo and its mother from the moment of conception, come into contact with this "downstream river" formed, first by oviductal fluid and then by uterine fluid. The MCU will act as if it were an "estuary", capturing part of this dialogue between mother and child that takes place in the oviduct and in the uterus. Although the sampling methods are varied, the MCU collection in a medical silicone cup that the woman can very easily insert into the vaginal canal up to the cervix ensures a sample with little or no contamination.

Our preliminary tests, with the installation of a silicone cup for 1 h, showed a variation of two times in the magnitude of the protein content during 10 consecutive days of collection, reaching a peak in the first four days after ovulation.

 

In conclusion, the MCU appears to be the ideal candidate to non-invasively track, capture and investigate biomarkers of embryonic development in vivo.

Cervicovaginal fluid

Various studies in the 1980s showed that immediately after fertilization, the embryo secretes a substance called “ovum factor”. Later in the 1990s, it was established that this factor corresponded to an embryonic form of platelet activating factor (PAF), a powerful phospholipid with multiple immunomodulatory actions, secreted by numerous types of cells in mammals [5]. PAF would be responsible, for example, for the moderate thrombocytopenia observed in women during pregnancy [14]; it is also linked to anaphylaxis [15].

 

However, the embryonic form of PAF is bound to albumin, which protects it from the action of PAF-AH, the enzyme responsible for the hydrolysis and inactivation of PAF; the embryonic albumin-PAF complex would distinguish it from free PAF and other forms of the molecule such as lyso-PAF. PAF has been confirmed as a fundamental and necessary molecule for the secretion of another factor called EPF (Early Pregnancy Factor), by granulosa cells in the ovary. EPF is an intracellular chaperone protein (chaperonin 10) that is surprisingly secreted from the beginning of pregnancy [16]. EPF appears 24 to 48 hours after conception and would be a predictor of the viability of the pregnancy. Recently, EPF has been established in cervico-uterine mucus (MCU) through activity in the rosetta inhibitory test (RIT). Interestingly, EPF activity disappears after an abortion, being undetectable after 24 hours. Its production is essential to protect the embryo from the mother's immune system and maintain pregnancy [17].

Another selected target is thioredoxin, an important redox modulator that makes it possible to defend cells from oxidative stress. Its importance is such that in the absence of its production, knockdown rat embryos for the expression of this protein are not able to survive and develop [18]. Its secretion has been demonstrated in culture of trophoblastic cells in vitro [12] although it has not been directly studied in the secretome of the human embryo, so it is unknown whether it would be a product released by it in vivo. However, its presence has been established in recent proteomics studies in cervicovaginal fluids from second trimester pregnant women [19]. If this protein is secreted by the embryo, the oviduct and / or the endometrium, it is plausible that its production and concentration increases by several orders of magnitude with early pregnancy, especially during the embryo's cell proliferation stages.

Another possible biomarker, leptin, has been identified as an essential protein in fertility studies in rats. Their absence makes them absolutely infertile; your fertility, however, can be restored by intrauterine injection of leptin. In vivo, the embryo would be exposed to leptin both in the oviduct and in the uterus, which would be almost entirely of maternal origin [20]. It has a fundamental role for the development and implantation of the blastocyst. As this protein is apparently not expressed under nonpregnant conditions in the female reproductive tract [21], it appears as a good target to be traced in MCU. Ubiquitin, an exclusively intracellular molecule for the physiological degradation of proteins, has surprisingly been detected as part of the secretome of human embryos (blastocysts) in vitro, placing it as another candidate to be traced in MCU. Another molecular target is the growth factor similar to the epidermal HB (HB-EGF), essential for the development and implantation of the blastocyst. A soluble form of this protein (sHB-EGF) appears involved in embryonic development, between the 8-cell stage and hatching (literally, this English word means “hatching”; it is the breaking of the zona pellucida that surrounds to the embryo so that it is in contact with the endometrium, during the beginning of implantation). Whether or not this protein is present in MCU in vivo has not been previously investigated.

 

Another biomarker, surprisingly not tracked in MCU, is human chorionic gonadotropin (hCG). Today hCG is used in all commercial pregnancy kits available, through its detection in urine several days or weeks after implantation. It is plausible that in MCU it is detected well in advance, since the embryo begins to produce it early before implantation, so that clear differences in concentration should be identified in this fluid. Finally, proteins known as histones H2A (in particular the ubiquitinylated histone, uH2A type 2C) participate in the process of silencing one of the X chromosomes in female embryos. Unlike male embryos that carry only one X chromosome, female embryos are required to completely silence one of these chromosomes from the first cell divisions, since the double gene dose would be harmful and potentially lethal [22]. In this way, its presence, if established in the MCU of women in the first days of pregnancy and absent both in negative controls and in controls established with a male pregnancy, would make it a useful marker to identify the sex of the embryo. Despite being a protein that is part of the nucleosome in adult cells and therefore canonically intracellular, the secretion of H2A has been demonstrated in neutrophils [23]; it has also been established in cervicovaginal fluid proteomes only from pregnant women [3, 24]. These characteristics make it an eligible and seductive target, since embryonic pluripotent cells could also secrete it, as they do with ubiquitin, thought to be an exclusively intracellular product.

 

Additionally, during the controlled "collision and blast" of the blastocyst when it makes contact with the uterine endometrium during implantation and invasion of the cytotrophoblast, embryonic cells (7 to 10 days from conception), DNA and epigenetic material is released into the uterine cavity. . We think, therefore, that it will be at this stage when uH2A can appear in MCU. The presence of embryonic trophoblastic cells in pregnancies of 7 weeks or more in brushings or exfoliates of the cervix has already been demonstrated and is being investigated as a diagnostic technique for the early screening of genetic abnormalities [25]. During the implantation window therefore, trophoblastic cells and various specific proteins could also accumulate in MCU and be traced in this fluid very early.

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