DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids

Infertility has become more common in many countries, particularly those where many people delay having children until later in life. To help individuals experiencing infertility conceive a child, scientists have developed treatments called assisted reproductive technologies (or ARTs for short). So far, more than 5 million children have been born with the help of these treatments. Most of the children seem healthy; however, birth defects are more common in ART-conceived babies than those conceived without treatment.

The cause of these birth defects is not known, though scientists suspect it may have something to do with techniques used in ART. One possible culprit is the liquid that is used in the laboratory to help the parents’ sperm and egg come together for fertilization. This same liquid is also used to bathe the developing embryo for the first few days after fertilization before it is implanted into its mother’s womb. Some scientists wonder whether adding the fluids normally found in the reproductive tract of their mother to this liquid could reduce defects in children conceived via ART.

Now, Canovas et al. have shown that fertilizing and growing pig embryos in liquids supplemented with fluid from the wombs of female pigs results in embryos that are closer to naturally conceived pig embryos than in non-supplemented liquids. In the experiments, naturally conceived embryos were compared to ART embryos exposed to the usual liquids and with ART embryos grown in liquids with fluid collected from the pig’s reproductive tract added. Cutting edge technologies were used to sequence the entire genomes of all of the embryos and compare which genes were active in each case. Canovas et al. also looked at chemical markers on the DNA – called epigenetic changes – that turn on or off the expression of genes without changing the DNA code itself.

The analysis showed that ART-conceived embryos grown in the usual liquid had different patterns of gene expression and epigenetic changes compared to naturally conceived embryos. Gene expression and epigenetic changes in the ART embryos grown with the pig reproductive fluid was more similar to the naturally conceived embryos.

These findings suggest that abnormal gene expression in the ART-liquid exposed embryos may lead to birth defects, and that using natural reproductive fluids may be safer. To confirm this, scientists will have to implant embryos conceived in these three different conditions into mother pigs and assess the health and gene expression patterns of the resulting piglets. If successful, these new insights might one day lead to improvements in ART techniques used to treat infertility in people.

“Most fertility researchers are trying to improve Assisted Reproductive Technologies (ARTs) success as measured by a single, clear standard: the birth of an apparently healthy baby. Only a few are trying to discern whether in vitro fertilization (IVF) leaves a subtle legacy in children. What will happen to these kids when they are middle-aged?” (Servick, 2014). In humans, according to a study by the World Health Organisation (WHO) in 190 countries, infertility affects 20% of couples and it was estimated that at least 40.5 million women were seeking infertility medical care in 2007 (Mascarenhas et al., 2012). ARTs provide a helpful alternative for a high proportion of infertility cases and the number of children born to date using these methods exceeds 5 million (International Committee for Monitoring Assisted Reproductive Technology I, 2012). Although the majority of them seem healthy, studies have reported higher rates of preterm births (Rubens et al., 2014), non-chromosomal birth defects and adverse perinatal effects in ART pregnancies (El Hajj and Haaf, 2013), with long-term effects being under study in humans (Kissin et al., 2014). Epidemiological data suggest that perturbed epigenetic gene regulation by the application of ART could be a contributory factor in these adverse outcomes (El Hajj and Haaf, 2013; Whitelaw et al., 2014), although such alterations could also be considered as consequences of parental characteristics, gamete quality or other non-epigenetic technique-derived effects (Simpson, 2014). To clarify the impact of each of these factors, the use of an animal model that avoids, as much as possible, the effect of parental circumstances and the use of protocols minimizing the technique-derived effects would help to attain the goal of offering safer ART for patients.

For modeling ART-related disorders in human, swine could be a good candidate for several reasons: their genetic, anatomical and physiological similarities with human (Swindle et al., 2012); their size and length of gestation; and the availability of individuals genetically selected by their excellent reproductive performance in artificial insemination centres. Importantly, this last trait could be useful to remove the paternal factor (low-quality male gametes) from studies as a possible reason for any epigenetic alterations found. However, most protocols for processing boar spermatozoa for IVF include their selection by density gradient centrifugations and just a few used the swim-up procedure to isolate highly motile spermatozoa which is the routine selection in human infertility clinics. Since it was observed that spermatozoa selected by swim-up show higher rates of normal morphology and motility, and decreased DNA fragmentation and methylation levels (Kim et al., 2015), it would be necessary to adapt the sperm selection protocols in pig before using them to model ART-derived epigenetic alterations.

In both mouse and human, accumulating evidence indicates that the embryo is sensitive to its very early environment and that culture media used in ART (as factors involved in technique-derived effects) may have long-lasting consequences (Kleijkers et al., 2014; Fernandez-Gonzalez et al., 2004). Several imprinting disorders and abnormal phenotypes have been linked to ART, but of special significance is the relationship between the presence of serum in culture media and the incidence of Large Offspring syndrome (LOS) in ruminants (Young et al., 1998), which includes diverse pathologic alterations and shows phenotypic and epigenetic similarities with the imprinted disorder Beckwith-Wiedemann syndrome (BWS) in humans (Chen et al., 2013). Since it was proposed that serum in the culture medium could be a crucial factor in LOS incidence, the tendency in the procedures for both human and livestock was to move toward the use of chemically defined media, limiting the presence of proteins in the culture medium to serum albumin. Although practical, this approach may have unpredictable consequences, because it ignores the fact that the reproductive fluids have a different composition to serum and are extremely rich in proteins other than serum albumin (more than 150 have been described in the oviductal fluid [Avilés et al., 2010]). If these proteins are physiologically present, they must play a variety of roles supporting the normal development of the embryo, roles that serum albumin alone cannot properly provide and serum cannot fully mimic. In addition, although ART in species such as cattle and sheep usually results in foetal overgrowth (Young et al., 2001; Chen et al., 2015), opposing phenotypes such as low birth weights (excluding BWS) are often seen in humans (Schieve et al., 2002) and pigs (García-Vázquez et al., 2010). A study showing the relationship between child birth weight and the protein source in embryo culture media (Zhu et al., 2014) reinforces the hypothesis that the protein composition of the culture media plays a role in the correct regulation of epigenetic marks in the growing embryo. A similar conclusion can be reached from a clinical trial showing that protein enrichment of media compared with addition of serum albumin alone improved the blastocyst implantation rate and may increase human births by more than 8% (Meintjes et al., 2009). Therefore, as with breast milk, which is so complex and so rich in bioactive factors that cannot be easily replaced with any artificial composition (Hennet and Borsig, 2016), the idea that reproductive secretions could be necessary in the culture media should not be underrated. At least, it should be explored under experimental conditions to unveil the relevance of these secretions.

DNA and RNA sequencing have become affordable cutting-edge technologies that could help to understand the mechanisms underlying abnormalities observed in ART-derived offspring. However, so far, single blastocyst whole-genome DNA methylation profiles comparing in vivo and in vitro produced embryos have not been published for any mammalian species and we therefore aim to produce these in this study.

We report here that modified swim-up protocols for the selection of spermatozoa in pigs and the use of reproductive secretions as additives in the culture media significantly increase the yield and quality of the blastocysts produced from a morphological, epigenetic and gene expression point of view. Using genome-wide analyses of gene expression by RNA-Seq and DNA methylation by Bisulfite-Seq in single blastocysts, we provide datasets of pig blastocysts produced in vitro with and without reproductive secretions as additives in the culture medium and show that the former are more similar to the in vivo specimens than the later. This suggests an alternative approach for conceiving healthier ART-derived children.

The milieu in which fertilization and embryo development takes place is crucial for healthy fetal and offspring growth, as revealed by developmental and epigenetic alterations as a consequence of in vitro culture and ART (Fernández-Gonzalez et al., 2004; Kleijkers et al., 2014; Lazaraviciute et al., 2014; Song et al., 2015). However, the progress made by ART during the past two decades make a future without their use inconceivable, thus it is necessary i) to characterize the real epigenetic cost of ART, separated from other factors and ii) to develop new protocols to safeguard against possible negative impacts in offspring. Our study evaluated, by single blastocyst profiling, the genetic and epigenetic impacts of modified protocols to produce embryos in vitro that mimic, as far as possible, the physiological conditions of fertilization and early embryo development. This imitation of the natural environment was first approached in both gametes separately: in the male gamete, by using sperm selection procedures that avoided centrifugations, and sperm washing and processing media containing oviductal fluid from the pre-ovulatory phase of the cycle; and, in the female gamete, by preincubating the oocytes within the precise fluid they encounter when, after ovulation, they are transported through the ampulla of the oviduct to the fertilization site, at the ampullar-isthmic junction (Halbert et al., 1988). Secondly, two experimental groups were established for a comparison with the in vivo specimens, where either BSA or reproductive fluids (obtained sequentially at the corresponding phases of the cycle) were added at every step of the IVF and EC procedures.

The results showed that reproductive fluids improve the outcome of IVF and the quality of pig blastocysts produced in vitro. The approach used, with spermatozoa coming from boars selected by their excellent reproductive performance, avoids the possibility of aberrations due to a paternal factor, which cannot be avoided in the human model, and helps to elucidate the epigenetic cost of ART independently of any paternal pathology. The figure of >40% progression of the cleaved embryos to blastocysts in vitro means an improvement over the best previous results (Redel et al., 2016). Nonetheless, the most remarkable findings were that Natur-IVF blastocysts attained a more advanced developmental stage and that the mean number of cells per blastocyst was the same as In-vivo embryos and 61% higher than C-IVF ones, which it is also above some of the best data previously reported in pigs (Redel et al., 2016). These results indicate that the use of reproductive fluids as additives, even at the low dose used in this study (1%) is beneficial for in vitro development of pig embryos so that it is now possible to obtain similar or even higher yields in the pig (45%) than in the bovine species. Although the possibility of transferring these methods to the human clinic might seem far off, the fact that nowadays other natural fluids such as breast milk for baby feeding or blood serum for transfusions are collected and stored at biobanks, make it possible to predict the future availability of human reproductive fluids obtained from oocyte donors during interventions at human infertility clinics (Coy and Yanagimachi, 2015). In fact, the first samples of these fluids are already stored at Biobanc-Mur in Spain (National Register of Biobanks N° B.0000859).

Our study also showed that Natur-ART blastocysts are closer to the gene expression profile of the In vivo blastocysts than C-IVF blastocysts. Amongst the most striking differences found was the expression of genes related to epigenetic reprogramming. It has been shown in mice and human that during the transition from zygote to blastocyst there is a massive loss of DNA methylation, with the exception of imprinted genes and some repetitive elements (Guo et al., 2014; Reik and Kelsey, 2014). In agreement with this observation, the global methylation level in the three groups of pig blastocysts analyzed was below 15%, suggesting that they had largely undergone a reprogramming event. This globally low methylation level compared to somatic cells or gametes, made it difficult to find high quantitative differences between embryos. Despite this, methylation percentage was higher in C-IVF embryos than in the other two groups, in agreement with previous studies indicating that ART-derived blastocysts displayed higher levels of methylation than in vivo derived ones (Deshmukh et al., 2011). This difference appeared to be global, with all features affected and, no evidence of multiple sub-groups over different genomic regions; therefore, there was no indication of specific regions resisting reprogramming. At the same time, genes for DNMT1 and the binding protein of its crucial cofactor UHRF1, which are considered responsible for maintenance of methylation patterns in replicating DNA and for maintaining imprints during preimplantation embryonic stages, were less expressed in C-IVF blastocysts, as was DNMT3B, required for de novo remethylation from this stage onwards. Differences in cell numbers, as a result of a probable additional round of cell division in In vivo and Natur-IVF embryos compared to C-IVF, is unlikely to explain a shift from ~11–12% to ~15% global methylation. All together, these data suggest an impaired demethylation in the C-IVF group. Analysis of hemimethylated CpG dyads by deep hairpin bisulfite sequencing, as recently reported in mouse (Arand et al., 2015), could help to clarify this issue.

A second key finding in this study was that the methylation levels in the samples analyzed showed much lower overall methylation levels (mean across all samples was 13.1%) than would be expected from somatic tissues. Furthermore, there were differences in the global mean methylation levels between different samples, ranging from 8.9% to 18.5%. Taken together, these observations suggest that the samples were collected during a time of global methylation reprogramming. The variability in global methylation levels would have confounded a direct comparison focussing on locus-specific methylation differences, so to account for this a quantile normalization was required to allow for a direct quantitative comparison of methylation levels.

Given that these samples are undergoing active reprogramming, it is also not unreasonable to think that some previously reported DMRs may not be established yet, or that the strength of the DMRs would be reduced. Despite this, we were able to find candidate DMRs between the groups with a reasonable statistical significance, although the magnitude of the methylation differences was low. Considering that previous studies have shown extremely close correlations between qPCR and RNA-seq data (Asmann et al., 2009; Griffith et al., 2010; Wu et al., 2014) and that validation by qPCR has its own probe-bias based on what region of the cDNA is amplified, we deem, in contrast to microarrays data, that there is not solid evidence that validation of the RNA-Seq and DNA methylation results by qPCR will provide extra significance to our results. For this reason, we did not perform qPCR validation in this study.

Another key observation in this study was that the in vitro culture affects imprinted gene expression and methylation. Plasticity of the preimplantation embryo could enable a recovery of alterations in methylation and further expression of non-imprinted genes during development, but any erosion of methylation marks at imprinted genes are unlikely to be corrected. In our data, from the 10 candidate imprinted regions retaining more than 30% of methylation in the pig blastocysts, we found three in C-IVF (ZAC1, PEG10 and NNAT) with significantly different methylation compared to In vivo blastocysts, and two (PEG10 and NNAT) compared to Natur-IVF. Knock-out mice lacking PEG10 showed early embryonic lethality with placental defects, indicating the importance of this gene in embryonic development (Ono et al., 2006). The protein encoded by NNAT, on the other hand, may be involved in the regulation of ion channels during brain development and may also play a role in forming and maintaining the structure of the nervous system. Defects in methylation at ZAC1 and IGF2R have been found in patients with the imprinted disorders transient neonatal diabetes mellitus (TNDM) or Silver-Russell syndrome (SRS), respectively, including those born following the use of ART (Le Bouc et al., 2010). In addition, genes related to the IGF axis, IGF2BP2 and IGF2BP2-IMP2, were up-regulated in C-IVF, and IGF2R in both C-IVF and Natur-IVF embryos. Altered IGF2BP2 expression in C-IVF is of interest, since reduced abundance of IGF2 has been associated with lower fetal weight after in vitro culture (El Hajj and Haaf, 2013). The imprinting status of IGF2R in the pig is unclear (Killian et al., 2001; Braunschweig, 2012) but, independently of this uncertainty, our data indicated higher expression of this gene in the two in vitro groups of blastocysts, which would be in agreement with previous reports in other species and could indicate a possibility of LOS-related alterations observed in abnormal in vitro and cloned embryos (Young et al., 2001). At the same time, the reduced methylation in IGF2R specifically in the C-IVF group could suggest that this group is more likely to be susceptible to sustained deregulation of IGF2R expression and a greater probability of LOS-like syndromes.

Altered expression in both groups of blastocysts produced under in vitro conditions was observed in some genes related to embryonic development, but some aberrations were absent in Natur-IVF embryos. In human blastocysts, it has been observed that those with higher implantation rate and higher number of cells per embryo showed up-regulation of DNMT3A (Kleijkers et al., 2015). In our data, the In vivo and Natur-IVF blastocysts showed a higher number of cells than those from the C-IVF group, in which expression of DNMT3A was decreased. We also observed higher expression of CDKN1A in the two in vitro groups, with an intermediate value in Natur-IVF. CDKN1A inhibits embryonic cell proliferation in response to DNA damage and it is considered one of the key genes responsible for the abnormalities in ART embryos since an aberrant increase of CDKN1A expression might be related to the growth-defect phenotype (Ishimura et al., 2016). Methylation of the CDKN1A gene, however, was similar in all three groups, between 5 and 7%. Other genes involved in DNA repair and cell cycle regulation were found to be altered, such as MDM2 (in C-IVF) and TP53INP (up-regulated in Natur-IVF and C-IVF) and HSPA4L, HSP40B1, HSPH1, HSP90 (down-regulated only in C-IVF). Altered expression of these genes may limit the ability of the embryo to respond to DNA damage, such that in vitro culture may lead to dysregulation of such genes, thus affecting long-term embryo viability (Zheng et al., 2005). The same situation was found for SLC2A3 (Glut-3) and SLC2A2, which have been related to LOS (Wrenzycki et al., 2004) and were highly up-regulated in the two in vitro groups. Again, no differences at the methylation level were found for any of these genes. Although DNA methylation at the promoter/gene bodies is directly/indirectly correlated with gene expression, this is not strictly true during the periods of dramatic loss of DNA methylation, as occurs during early embryo development or primordial germ cells (PGC) formation. For example, Gkountela et al. (2015) showed a general uncoupling between DNA methylation and gene expression during demethylation of PGCs, commenting ‘Our data reveal a remarkable and pervasive loss of DNA methylation in human PGCs and AGCs during prenatal life that has almost no relationship to changes in gene expression’. Comparative analyses between our methylation and gene expression data also showed this lack of correlation. In our opinion, at this stage of development and with this low level of methylation, this was an expected result.

Finally, the exclusive alteration in C-IVF of genes such as KIT, whose knock-out in mouse results in multiple alterations including embryonic lethality (Ro et al., 2010), UBR2, whose deletion results in female embryonic lethality and growth arrest (Kwon et al., 2003), or ISOC1, whose mutation produces phenotypes with body weight loss (Rainger et al., 2013), support the hypothesis that offspring produced with Natur-IVF conditions would be healthier than those produced with C-IVF, although additional studies are necessary to confirm this finding.

In conclusion, we report here the first time genome-wide DNA methylation and transcription analysis in single blastocysts (in vivo and in vitro) of a mammalian species and propose a new strategy for prevention of aberrant epigenetic and gene expression profiles induced by ART. This strategy, based on the addition of reproductive fluids in the culture media used during the ART procedures, can be applied in other animals as well as in humans, after safety concerns of transmission of diseases have been properly addressed. The design of new culture media containing all the proteins that are naturally present in the original biological fluid, represents not only a technical challenge but a biomedical responsibility that must be addressed to prevent future pathologies both in animals and humans. In addition, we offer a new protocol for the in vitro production of pig embryos with a significant improvement over the previous data published. Our study represents a new form of thinking in the field, far from the chemically defined culture media, and could help to face one of the biggest milestones of the current reproductive medicine: safer ART.

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Author details

1. Sebastian Canovas

   1. Physiology of Reproduction Group, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia-Campus Mare Nostrum, Murcia, Spain
   2. Instituto Murciano de Investigación Biosanitaria, Murcia, Spain

   Contribution

   SC, Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-4190-6258

2. Elena Ivanova

   Epigenetics Programme, The Babraham Institute, Cambridge, United Kingdom

   Contribution

   EI, Data curation, Validation, Methodology, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

3. Raquel Romar

   1. Physiology of Reproduction Group, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia-Campus Mare Nostrum, Murcia, Spain
   2. Instituto Murciano de Investigación Biosanitaria, Murcia, Spain

   Contribution

   RR, Data curation, Validation, Investigation, Methodology, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

4. Soledad García-Martínez

   1. Physiology of Reproduction Group, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia-Campus Mare Nostrum, Murcia, Spain
   2. Instituto Murciano de Investigación Biosanitaria, Murcia, Spain

   Contribution

   SG-M, Data curation, Methodology

   Competing interests

   The authors declare that no competing interests exist.

5. Cristina Soriano-Úbeda

   1. Physiology of Reproduction Group, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia-Campus Mare Nostrum, Murcia, Spain
   2. Instituto Murciano de Investigación Biosanitaria, Murcia, Spain

   Contribution

   CS-Ú, Data curation, Methodology

   Competing interests

   The authors declare that no competing interests exist.

6. Francisco A García-Vázquez

   1. Physiology of Reproduction Group, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia-Campus Mare Nostrum, Murcia, Spain
   2. Instituto Murciano de Investigación Biosanitaria, Murcia, Spain

   Contribution

   FAG-V, Data curation, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

7. Heba Saadeh

   1. Epigenetics Programme, The Babraham Institute, Cambridge, United Kingdom
   2. Bioinformatics Group, The Babraham Institute, Cambridge, United Kingdom

   Present address

   Computer Science Department,KASIT, The University of Jordan, Amman, Jordan

   Contribution

   HS, Data curation, Methodology

   Competing interests

   The authors declare that no competing interests exist.

8. Simon Andrews

   Bioinformatics Group, The Babraham Institute, Cambridge, United Kingdom

   Contribution

   SA, Software, Formal analysis, Validation, Methodology, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

9. Gavin Kelsey

   1. Epigenetics Programme, The Babraham Institute, Cambridge, United Kingdom
   2. Centre for Trophoblast Research, University of Cambridge, Cambridge, United Kingdom

   Contribution

   GK, Supervision, Funding acquisition, Methodology, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-9762-5634

10. Pilar Coy

    1. Physiology of Reproduction Group, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia-Campus Mare Nostrum, Murcia, Spain
    2. Instituto Murciano de Investigación Biosanitaria, Murcia, Spain

    Contribution

    PC, Conceptualization, Data curation, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Writing—review and editing

    For correspondence

    pcoy@um.es

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0002-3943-1890

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