MiR-191-5p is upregulated in culture media of implanted human embryo on day fifth of development

Background Morphological features are the most common criteria used to select human embryos for transfer to a receptive uterine cavity. However, such characteristics are not valid for embryos in cellular arrest. Even aneuploid embryos can have normal morphology, and some euploid embryos have aberrant morphology. The aim of this study was to quantify the expression profile of hsa-miR-21-3p, -24-1-5p, -191-5p, and -372-5p in culture media on day 5 of in vitro embryo development, and compare the profiles of two groups of media classified by outcome: successful (n = 25) or unsuccessful (n = 25) implantation pregnancy. Methods Fifty patients were accepted in the Department of Reproductive Biology of a Hospital in México City, based on the Institutional inclusion criteria for in vitro fertilization. Embryos were transferred to the women on day 5 of cultivation, and the culture media were collected. RNA was isolated from each culture medium with TRIzol reagent, and microRNA (miRNA) expression was detected through RT-PCR with specific primers. Expression bands were quantified by reading optical density. Results There was a 5.2-fold greater expression of hsa-miR-191-5p in the pregnancy-related culture media (p ≤ 0.001) and a 1.6-fold greater level of hsa-miR-24-1-5p (p = 0.043) in the media corresponding to non-pregnant women. No significant difference existed between the two groups hsa-miR-21-3p (p = 0.38) or hsa-miR-372-5p (p = 0.41). Conclusions Regarding adequate in vitro embryo development, hsa-miR-191-5p could possibly represent a positive biomarker, while has-miR-24-1-5p may indicate poor prognosis. This former miRNA modulates IGF2BP-1 and IGF2R, associated with the implantation window. On the other hand, hsa-miR-24-1-5p may be related to a poor prognosis of human embryo development.


Background
MicroRNAs (miRNAs) are a large class of small noncoding RNAs approximately 23 nucleotides in length. Their important functions in post-transcriptional gene expression are carried out by binding to the complementary sequence of the 3′untranslated region (3′-UTR) or by degrading target messenger RNA (mRNA) transcripts via complementary base pairing [1]. MiRNAs are essential to many cellular processes and can be transferred between cells as a mode of cell-cell communication [2] and are relatively stable when circulating [3].
The aim of the current study was to quantify the expression profile of hsa-miR-21-3p, -24-1-5p, -191-5p, and -372-5p in culture media on day 5 in vitro fertilization (IVF) embryo development, and compare the profiles of two groups pf media classified by outcome: successful (n = 25), or unsuccessful (n = 25) implantation and pregnancy in patients.

Ethics approval
Embryos were cultured in media and transferred to women on the fifth day of development. On this same day, the media were collected for analysis. The current protocol was approved by the Ethics and Research Committees of the Instituto Nacional de Perinatología, Ciudad de México, México (212250-22661). The prospective participants received an explanation of the purpose of the present investigation, and those willing to take part signed informed consent. All candidates signed the consent form.

Patient selection
The study was conducted on a total of 50 female patients who received a diagnosis of infertility and requested in vitro fertilization (IVF). The inclusion criteria were the following: ≤37 years of age, regular menstrual cycles, normal uterine cavity confirmed by hysteroscopy, the absence of intrauterine adhesion or inflammation, ≥7 mm endometrial thickness in the late follicular phase (determined by ultrasonography), a normal ovarian reserve (<9.0 mU/mL follicle-stimulating hormone), a normal ovarian response to the stimulation protocols (>8 oocytes retrieved in a controlled ovary hyperstimulation cycle), and no use of exogenous hormone (estradiol/progesterone) during the endometrial cycle. The exclusion criterion was the failure of the woman to undergo an ultrasound scan within 4 weeks after a positive pregnancy test. Non-inclusion criteria were endometrial cancer or hyperplasia, endometriosis, and having a male partner with infertility.

Ovarian stimulation
The patients received controlled ovarian stimulation based on an assessment of FSH/LH. Upon observing a follicular cell greater than 18 mm in diameter, the ovaries were stimulated with chorionic gonadotropin (hCG, Merck Serono, Switzerland). Mature oocyte were captured with ultrasound guidance 36 h posterior to hCG injection, and IVF was performed on day 5 after oocyte retrieval.

In vitro fertilization
Fertilization of oocytes was carried out in vitro with capacitated sperm (1 × 10 6 cells/mL) and evaluated by the presence of a second polar body. The progression of cell division was monitored daily until it reached the 36-cell stage. Successfully fertilized oocytes were individually maintained in 25 μL of G-1 PLUS culture medium (Vitrolife Sweden AB, Sweden). The embryos were cultured in a ASTEC incubator (EC6S-MD, USA, PA) at 37°C with 5% O 2 /6% CO 2 until being transferred to women on day 5.
Two embryos with type I, II, or III quality on the third day of embryonic development were transferred to the uterine cavity during the implantation window using the Soft Cook technique and Flexible Pass intrauterine transfer cannula guided by an abdominal ultrasound apparatus equipped with a real-time, 5-MHz sector electronic array endovaginal probe (Philips Epiq CVx; MO, USA).
Fourteen days after embryo transfer, ultrasound was employed to examine the possibility of successful implantation, evidenced by the development of the embryonic sac. Based on the results, the patients were assigned to one of two groups: (1) implanted embryos (n = 25, pregnant patients) and (2) non-implanted embryos (n = 25, non-pregnant patients).

RNA isolation, cDNA synthesis, and PCR-reaction
The culture medium was obtained on day 5 of embryonic development, and total RNA was extracted with the TRIzol reagent (InvitroGen, Carlsbad, CA), according to the manufacturer´s instructions. The concentration of RNA in each sample was measured as the A 260 /A 280 ratio on a NanoDrop One spectrophotometer (Thermo Scientific, Waltham, MA, USA).
Twenty μL of PCR amplicons were mixed with Tris/ Acetic Acid EDTA 1X loading buffer (Bio-Rad, Hercules, CA, USA) and added to wells containing 4.0% agarose gel. Electrophoresis was then run at 60 V at a constant temperature for 40 min.
Subsequently, the gel in each well was visualized and the image captured by a UV transillumination system (Gel Doc 2000, Bio-Rad, Hercules, CA, USA). The band of expression for each miRNA of interest was identified by optical density with the ImageJ program (NIH, USA).

Blood samples
Five milliliters of peripheral blood were obtained from the patients by puncture of the cephalic vein, placed in EDTA-K2 tubes (BD Vacutainer), and centrifuged at 14, 000 rpm for 10 min. Serum was collected in Eppendorf tubes and stored at −70°C until to quantification hormones assay.

Assay to determine the levels of sex hormones
All evaluations of hormones were performed in the central laboratory of the Instituto Nacional de Perinatología on the Modular Analytical apparatus cobas e 411 (Roche, USA). A commercially available assay kit was used to measure the serum levels of P4, E2, T4, FSH, LH, AMH, and hCG (Roche system, USA), with according to the manufacturer´s recommendations and as previously described [27][28][29]. The lower limit of detection for these hormones was 0.4 ng/mL, 5 pg/mL, 0.025 pg/ mL, 0.100 mIU/mL, 0.100 mIU/mL, and 0.2 ng/mL, 0.1 mIU/mL, respectively. The intra-assay coefficient of variation was 3%, 5%, 5%, 3%, 2%, 3%, and 5% respectively.

Statistical analysis.
Significant differences between the two groups of women (those with implanted or non-implanted embryos) were determined in relation to patient characteristics, hormonal concentrations, and expression of miRNAs.
Data are expressed as the mean ± SD and examined with Student's t-test on SigmaStat version 4.0 (Jandel Co., CA, USA), considering p < 0.05 as statistically significant.

Patient characteristics
The clinical characteristics of pregnant and nonpregnant patients were show in Table 1. There were no significant differences between the two groups in regard to mean age (p = 0.23), body mass index (p = 0.43), length of the menstrual cycle (p = 0.71), duration of menstruation (p = 0.54), or endometrial thickness on the day of the LH surge (p = 0.45).

Discussion
Although embryonic morphology is the criterion used in clinical practice to evaluate the viability of embryos for transfer to patients [30,31], this method selection has not demonstrated an acceptable correlation, with outcome. Even aneuploid embryos can have normal morphology, and some euploid embryos can have aberrant morphology [32]. Among the various proteins reported as possible biomarkers for embryonic development [31,33], none have yet proven to be good indicators of a successful implantation in the receptive endometrium.
During the development of murine embryos from the first stages of cell division to the formation of blastocysts, expression of miRNAs predominates over other non-coding RNAs, suggesting their likely role in regulating distinct pathways of differentiation and cell proliferation [34]. In the current study, the comparation of the expression of four miRNAs between the two groups of embryo culture media (corresponding to pregnant versus non-pregnant patients) revealed important differences. There was a stronger expression of hsa-miR-191-5p in the culture media of embryos leading to pregnancy (p ≤ 0.001), and of hsa-miR-24-1-5p in the samples corresponding to non-pregnant patients (p = 0.043). The miR-NAs that showed no significant difference between two groups were hsa-miR-21-3p and hsa-miR-372-5p (Fig. 1C).
The activity of hsa-miR191-5p on endometrial markers in the implantation window and of hsa-miR-24-1-5p on cell proliferation and migration is represented in a model (Fig. 2). Rosenbluth et al., (2014) described an increase in miR-191 in the culture media of developing embryos having undergone implantation [35]. The current results indicate a significant 5.2-fold greater expression of this miRNA in the culture media of embryos of pregnant versus non-pregnant patients. Recently Wang et al., (2016) reported that the expression of hsa-miR-191-5p modulates various proteins, two of which belong to the insulin-type growth factor family (IGF2BP-1 and IGF2R), associated with the decidualization of endometrial tissue [36]. According to the data in the literature, miRNAs, are not only potential biomarkers of implantation feasibility, but also could be secreted to the  Data are presented as the mean ± standard deviation extracellular environment to induce activation of the cells or white tissues favoring embryo implantation and embryonic development. Kropp et al., (2015) transfected morula stage embryos with a mimic of miR-24. When they progressed to the blastocyst stage, there was morphological damage (compared to the control group), apparently related the activation of cellular apoptosis [20]. The authors demonstrated that one of the target mRNAs of miR-24 is CDKN1b, a cell cycle regulator found to be significantly reduced in the experimental control group [20]. Moreover, an elevated expression of hsa-miR-24 has been linked to the suppression of osteoblast differentiation [37], failure of embryo implantation into the endometrium [38], and adverse obstetrics tests results related to preeclampsia and intrauterine growth restriction [38,39]. Thus, the current 1.6-fold greater expression of has-miR-24-5-p in the culture media of embryo of nonpregnant versus pregnant patients is in agreement with previous reports (Fig. 1C).
The two miRNAs with a lack of significant difference between groups in the present study hsa-miR-21-3p and hsa-miR-372-5p are both "constitutive", according to an analysis by prediction software. They are involved in the regulation of MAP3K-1 and CDK6 cyclin, which are critical genes in the cell cycle as well as the signaling and apoptosis pathways [40]. MiR-372-5p is known to aid in the conversion of human fibroblasts into pluripotential stem cells, suggesting a crucial role of this miRNA in balancing differentiation and the maintenance of cell pluripotency.
Consequently, some miRNAs are linked to embryonic viability and others to poor prognosis. In the future, the Fig. 1 Expression of hsa-miRNAs in the culture medium of human embryos with type II development. Marker (Lane 1), negative control (Lane 2) samples of the culture medium of the 25 implanted (A, pregnant patients) and non-implanted embryos (B non-pregnant patients). The optical density of each band was determined and the mean ± standard deviation is shown (C). The significant difference is indicated which was made by the Student's t-test and was taken as a difference of less than 0.5 full description of embryonic miRNome may be an especially useful tool in the clinical field.

Conclusions
The was a 5.2-fold greater expression of has-miR191-5p in the culture media corresponding to pregnancy and a 1.6-fold higher level of has-miR-24-1-5p in the media related to non-pregnant patients. Hsa-miR-191-5p could possibly serve as an invaluable biomarker of human embryo development, while has-miR-24-1-5p may be an indicator of poor prognosis of the same. The former miRNA modulates two proteins, IGF2BP-1 and IGF2R, to be associated with the implantation window. Our research group is currently evaluating the participation of has-miR-191-5p in the regulation of proteins associated with embryo implantation and in the activation of other miRNAs after the transfection of endometrial cells. In the future, the full description of the embryonic miRNA genome might be a very useful tool in the clinical field.
Abbreviations BMI: body mass index; CDK6: cyclin-dependent kinase 6; IVF: in vitro fertilization; hsa-miR: homo sapiens-microRNA; PCR: polymerase chain reaction; RT: retrotranscription; MAP3K-1: mitogen-activated protein kinase Acknowledgments This project is part of the experimental work of Ricardo J. Acuña-González to obtain the degree of Master of Science (514220750) from the Programa de Ciencias Médicas, Odontológicas y de la Salud, Universidad Nacional Autónoma de México (UNAM). We thank CONACyT for providing financial support for this study.
Authors' contributions RJAG: obtained the culture medium for the development of the embryos, performed in vitro fertilization, and determined miRNA expression by optical density. MOV: was responsible for the quantification of the hormones in patient serum. MOC: monitored the patients with ultrasound imaging. RJAG and MOV carried out the experiments for RNA isolation, and RT-PCR. RJAG, MOV, JSLC, JLC, and HFH participated in the analysis and discussion of results. RJAG, MOC and HFH were in charge of the writing of manuscript. HFH designed the study and obtained financial support.

Funding
The current study was supported by a grant (212250-22661 assigned to HFH) from the Instituto Nacional de Perinatología "Isidro Espinosa de lo Reyes" of Ciudad de México, México. The institute was not involved in any stage, and therefore has no conflict of interest with the content of the manuscript.

Availability of data and materials
All of the relevant information from the study is described in the manuscript.

Declarations
Ethics approval and informed consent Each patient was informed that, by agreeing to participate in the study, the culture medium of the embryo would be used to perform miRNA expression assays after embryo transfer, and that this procedure would not affect the development of the embryos. All candidates signed the consent form after the explication. The present protocol was reviewed and approved by the Ethics and Research Committees of the Instituto Nacional de Perinatología (212250-22661).

Fig. 2
Differential expression of hsa-miR-191-5p and hsa-miR-24-1-5p in the culture medium of human embryos with type II development. The secretion of hsa-miR-191-5p by developing embryos stimulates the expression of insulin-like growth factor-associated proteins (IGF2BP-1 and IGF2R) associated with the implantation window in endometrial cells and responsible for inducing major changes in the decidualization of endometrial tissue (A) [14]. For its part, the increase in the expression of hsa-miR-24-1-5p in the culture medium of developing embryos of non-implanted (non-pregnant patients) has been associated with inhibition in cell proliferation and migration (B) Received: 27 November 2020 Accepted: 12 May 2021