Influence of conception and delivery mode on stress response marker Oct4B1 and imprinted gene expression related to embryo development: A cohort study

Abstract Background Recent scientific data support that the mode of conception and delivery may influence epigenetic regulation and therefore embryo development. Octamer-binding transcription factor 4-B1 (OCT4B1), a novel variant of OCT4 with yet unknown biological function, is suggested to have a potential role in mediating cellular stress response. Furthermore, Insulinlike Growth Factor 2 (IGF2), Mesoderm-specific Transcript (MEST) and paternally expressed gene 10 (PEG10) are genes known as imprinted and are regulated via means of epigenetic regulation. The influence of delivery mode and conception on epigenetic regulation is an active research field. Objective Our aim was to correlate the expression level of Oct4B1 and the expression and methylation level of IGF2, MEST, and PEG10 imprinted genes with the mode of delivery and conception in the umbilical cord blood of newborns. Materials and Methods Samples of umbilical cord blood from infants born after vaginal delivery, caesarean section (CS) with the infant in cephalic position and CS due to breech position were examined. Furthermore, the investigation included infants conceived through means of assisted reproductive technology. Results No statistically significant differences were found in mRNA expression levels between different modes of conception and delivery (p = 0.96). Oct4B1, IGF2, MEST, and PEG10 expression levels do not seem to be significantly affected by different modes of conception and delivery. Conclusion These results indicate that the expression and methylation patterns of Oct4B1, IGF2, MEST and PEG10 in umbilical cord blood are not affected by the conception and delivery mode.


Introduction
Vaginal delivery (VD) exposes infants to various stressors absent during caesarean section (CS), such as the secretion of catecholamines and cortisol, which trigger hormonal cascades in order to prepare the infant for life after birth (1). Furthermore, the artificial interventions employed during assisted reproductive technologies (ARTs), coincide with the embryos' epigenetic reprogramming, thus leading possibly to aberrant establishment and maintenance of genomic imprints and an increase in imprinting disorders (2)(3)(4)(5). On the other hand, concerning cellular stress, the octamer-binding transcription factor 4-B1 isoform, (Oct4B1), has emerged as a possible cell stress marker (6). This isoform has been found to be an emergent marker of stemness in embryonic cells and is also implicated in apoptosis and probably in cancer (7)(8)(9)(10). In the present study, Oct4B1 was selected to be studied because of its putative role in cellular stress response. This is the first study conducted in umbilical cord blood (UCB) concerning Oct4B1. Other genes susceptible to epigenetic modification upon stress exposure are Insulin Growth Factor 2 (IGF2), Mesoderm-Specific Transcript (MEST), and Paternally Expressed Gene 10 (PEG10). These genes affect fetal growth and their disruption is linked to metabolic disorders, cognitive impairment, low birth weight, and some types of cancer (11)(12)(13). These genes were selected to be studied because of their crucial role in fetal development, as they control embryonic and placental growth. Furthermore, these imprinted genes are responsive to different in utero environments and thus may mediate adverse environmental signals to the embryo during pregnancy. There are contradictory results in literature concerning the effect of ART on these genes. Lastly, there are no previous reports of the expression of these genes in embryos with breech presentation.
Given that various environmental factors and stressors can affect the epigenetic processes, concerns are raised about the implications of the increasing prevalence in elective CS deliveries and the use of ART. Our aim was to investigate the role of different modes of conception and delivery as possible environmental stressors on epigenetic regulation and stress response. Therefore, we evaluated the expression level of Oct4B1 and the expression and methylation status of IGF2, MEST, and PEG10 imprinted genes in UCB samples of newborns.

Sampling method
In this cohort study, 40 participants were enrolled whose UCB samples were collected during the time of their delivery. The samples were subdivided into four groups that comprised of 10 participants each. The extremely strict inclusion criteria did not allow for a larger sample size to be collected. This may be a disadvantage for the statistical analysis of the results, but it reflects a very homogenous and carefully designed sample population. The groups examined were VD (control group), CS with cephalic projection of the embryo, and CS with breech embryo projection. Additionally, the fourth group consisted of infants conceived through ART and delivered through caesarian section.
The eligibility for participation included uncomplicated full-term deliveries (≥ 37 wk) of healthy parents with an existing full medical record and supervision of the pregnancy by an obstetrician. On the other hand, the exclusion criteria included positive smoking status, chronic or acute diseases, twin gestations, non-compliance to the required medical tests during pregnancy, and pregnancies with embryos presenting chromosomal or anatomical abnormalities. All biospecimens collected were done so with the participant's informed written consent after having secured permission from the Scientific Committee of Papageorgiou General Hospital of Thessaloniki, the Bioethics Committee of the School of Medicine of Aristotle University of Thessaloniki, and from the Hellenic Data Protection Authority.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from leucocytes using TRIZOL reagent (Invitrogen, USA), according to the manufacturer's instructions. RNA samples were treated with TURBO DNA-free TM DNase (Ambion, Life Technologies, USA). A no-reverse transcription (no-RT) control was used for each sample to exclude any potential non-specific amplification of genomic DNA. The SuperScript TM First-Strand Synthesis System (Invitrogen, USA) was used to reverse transcribe total RNA with random hexamer primers. Quantitative Real-Time PCR employed TaqMan TM Gene Expression MasterMix reagent (Thermo Scientific, USA) and TaqMan TM Gene Expression Assays for evaluation of Beta-2 Microglobulin (B2M, endogenous control) IGF2, MEST, and PEG10 transcription. The primers for Oct4B1 were previously described ( Table I). Quantification of the results was performed using the 2 −ΔΔCt method after normalization against B2M.

Methylation-specific PCR
For DNA extraction, DNA Mini Kit (Qiagen, Netherlands) was used. DNA recovered from the aforementioned process was chemically converted using EpiTectPlus DNA Bisulfite Kit (Qiagen, Netherlands), in order to turn unmethylated cytosines to uracil for detection by methylation-specific PCR. Next, the bisulfitetreated DNA was subjected to PCR for detection of the methylated and unmethylated MEST, IGF2, and PEG10 alleles (Table II). Samples containing no DNA were used as a negative control, whereas for positive control, bisulfitetreated DNA from peripheral blood was used.

Ethical considerations
All procedures performed in studies involving human participants were in accordance with the ethical standards of the Scientific Committee of Papa Georgiou General Hospital of Thessaloniki (reference number: 157/27.06.12), the Bioethics Committee of the School of Medicine of Aristotle University of Thessaloniki (reference number: 1/8-11-2012), and of the Hellenic Data Protection Authority (reference number: 1145) and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. In addition, a written informed consent was obtained from individual participants included in the study.

Statistical analysis
Data were analyzed using the IBM SPSS Statistics version.24 (SPSS Inc., Chicago, Illinois, USA) program. Demographic data were normally distributed (based on Shapiro test for normality) and one-way ANOVA-test was used for quantitative variables (maternal age, maternal weight, gestational week, and birth weight) and χ 2 -test for qualitative variables (gender of the newborns and ratio of primiparae women). The gene expression results were evaluated through non-parametrical tests due to the small sample size, and more specifically the Kruskal-Wallis test was employed to compare Oct4B1, IGF2, MEST, and PEG10 expression between the four groups. Results were deemed significant only when p < 0.05.

Results
As mentioned above, 40 participants were enrolled whose UCB samples were collected during the time of their delivery (Table III). Quantitative Real-time PCR revealed no statistically significant differences at the transcriptional level between the VD, CS, and ART groups for Oct4B1 (p = 0.58), IGF2 (p = 0.96), MEST (p = 0.75), and PEG10 (p = 0.37) (Figure 1). PEG10 exhibited a nonsignificant trend for up regulation (p = 0.089) in the group of cephalic presentation CS compared to the VD group. Methylation-Specific PCR products were subjected to gel electrophoresis in order to validate the presence of both methylated and unmethylated alleles for IGF2, MEST, and PEG10 for all 40 samples (Figure 2). Methylation analysis was not possible for Oct4B1 due to the existence of alternative promoters for this isoform, incommoding primer design (16). Methylation status is in agreement with non-statistical differences in expression.

Discussion
It is assumed that imprinted genes can serve as environmental sensors, since they can be responsive to environmental stressors and alter their expression and/or methylation during specific developmental stages.
In the current study, we investigated the effect of the conception and delivery mode, which are This theory was first described in animal reports (20,21), but many scientists believe that it also refers to humans (22)(23)(24)

Conclusion
The