Oh Baby!

Analyzing the effects assisted reproductive technologies (ART) have on women with DMNT-10 deficiency and the developmental process of their offspring

By: Ellen Saurer

Edited By: Grace Owens-Kurtz, Namrata Damle, Madeline Nicol, and Katherine Hill

Image Credit: Maurizio De Angelis/Wellcome Images

In a world where an estimated 72.4 million couples globally experience infertility, assisted reproductive technologies, otherwise known as ARTs, are vital to fulfill the wish of having a baby. About 1–6% of all live births in developed countries are conceived through ART and living life thanks to ART’s. I’m one of them. So is my twin sister.

I’ve always grown up wondering exactly why my twin and I looked so different. She had curly hair and bright blue eyes while I had straight hair and green eyes. It was only a couple years ago that I found out why this was. While there are many fraternal twins that are born naturally, I found out that I am an in-vitro baby, or as I started joking, a “test-tube baby”.

Science is always changing by expanding knowledge and new discoveries. Today, genetics especially is becoming a part of daily life. To name just one example, genetic counselors can take your family history to determine your risk for developing cancer get or find the probability of your offspring being born with a genetic disorder. Because this information is now so available, it’s getting utilized more. ART has become a reliable option for families that struggle with infertility or have a history of genetic disorders. While ART can seem perfect, there are still many flaws, and as ART continues to gain popularity, it’s important to understand the consequential effects and, specifically the effects ART has on women with DMNT-10 deficiency — an enzyme important to the development of offspring — and on their children’s developmental process

These flaws are represented in the scientific research paper, Compromised Oocyte Quality and Assisted Reproduction Contribute to Sex-Specific Effects on Offspring Outcomes and Epigenetic Patterning, where the question remains on whether defects observed in ART-conceived children are due to the ART techniques themselves, to factors related to underlying infertility, or to an interaction of the two.

The are a wide variety of assisted reproductive technologies, which include, but are not limited to:

1. In-vitro fertilization (IVF), when sperm and egg combine and fertilize outside the body and the embryo develops for three to five days before being transferred into the woman’s uterus,
2. Zygote intrafallopian transfer (ZIFT), when sperm and egg fertilize outside of the body and are then inserted inside the woman’s fallopian tubes using a catheter (see image), and
3. Gamete intrafallopian transfer (GIFT), which involves immediately transferring mixed sperm and egg into the fallopian tubes, which are able to fertilize once in the body.

In all techniques, ART requires the collection of eggs from a woman and the collection of sperm from a man to be combined outside the body, typically in a test tube in the hopes of fertilization, and inserted back into either the fallopian tube, where fertilization naturally occurs, or inserted inside the uterus so the already fertilized embryo can attach to the uterine wall. Of all of the techniques available, IVF is the most well-known and performed, as about 99 percent of ART procedures done in the United States are IVF treatments (Ding, 2016). While it’s important to understand how ARTs work, it’s also crucial to understand exactly why a woman may have infertility problems.

While there are varying reasons as to why a woman may struggle with infertility, some may have deficiencies with their enzymes specific to the oocyte. An oocyte is a cell in an ovary that carries the potential to form an ovum, or immature egg. The oocyte-specific enzyme DNMT-10 plays a critical role in preimplantation development. In a recent study looking at DNMT-10 levels in women, some women had a 50 percent reduction in their DNMT-10 levels found in their oocytes compared to women who had typical DNMT-10 levels; those with the 50 percent reduction were more prone to participate in ART while, for the most part, women with typical DNMT-10 levels did not require ART, also known as Naturally Cycling (NAT). The subjects with reduced DNMT-10 levels were thus classified as having a DNMT-10 deficiency.

The study then looked into how DNMT-10 deficiency may affect egg cell viability and attachment. Eggs were collected from NAT individuals and DNMT-10 deficient individuals. Women with DNMT-10 deficiency have a higher chance of their eggs dying around mid-gestation, and these results showed that there was an increase in post-implantation loss in the DNMT-10 deficient woman, from five percent in NAT women to 19 in DNMT-10 deficient women.

Defects were also observed in craniofacial abnormalities — birth defects of the face or head (see picture), and in neural tube formation in the developing fetuses when combined with ART. Women with DNMT-10 deficiencies had an increased rate of morphological, or structural, abnormalities in their children (2.5 percent in women without the deficiency compared to 17.9 percent in DNMT-10 deficient women). Strangely, these abnormalities were only identified in males, though there was no significant difference in sex ratios between the two groups observed (67.6 percent males in the NAT group versus 55.6 percent males in DNMT-10 group). Still, these results highlight that in combination with ART, DNMT-10 deficient women had increased rates of embryonic abnormalities in males mid-gestation and that DNMT-10 deficiency alone disrupts normal embryo development in the post-implantation period.

Craniofacial abnormalities — picture includes cleft lip and palate, which is the most common birth defect (MedlinePlus)

Researchers then looked at the DNA methylation patterns present in DNMT-10 deficient women. DNA methylation silences gene expression — the process by which information within a gene is made into a functional product, such as a protein — by preventing the production of functional proteins. Methylation is a type of epigenetic inheritance, meaning that it is a type of change that is heritable, but does not actually alter the DNA sequence itself. DNMT-10 deficiency in the NAT group created changes in DNA methylation patterning similarly in males and females but did not result in an increase in embryo or placental abnormalities or sex-specific effects. It was only when DNMT-10 deficiency and ART were combined that an increase in embryo abnormalities and sex-specific effects were found. In ART-conceived children, an increase in rare genomic imprinting disorders were observed, including Beckwith-Wiedemann (see image) and Angelman Syndromes (AS). Genomic imprinting is a type of epigenetic inheritance deviance when one set of a parent’s genes are silenced and plays an important role in normal mammalian growth, development, and placenta functions, cancers, and abnormal neurobehavioral development.

This study reveals an interaction between subfertility (condition of being less than normally fertile, but still fertile), and commonly used ART techniques. These changes in DNA methylation are likely to have functional consequences on the placenta, affecting normal offspring growth and development. Additionally, sex-specific effects were also found when DNMT-10 levels were decreased, but not absent, in oocytes; a condition more likely to be associated with female subfertility (as an effect of age). This study further supports the hypothesis that the interaction between subfertility and ART may likely play a role in mediating poor embryonic outcome.

Reduced Representation Bisulfite Sequencing (RRBS) is a technique that sequences only a reduced, representative sample of the whole genome (Lee, 2014). Following ART, RRBS revealed sex-specific DNA methylation changes across the entire genome with an increased placental sensitivity to DNMT-10 deficiency when compared to ART alone. Ultimately, this data revealed the placenta to be more sensitive to epigenetic deviance, an observation that is in the same with several other ART studies in mice.

Epigenetic deviations in the placenta may result in impaired function and follow harmful effects on the embryo. Specifically, in the ART female placenta, the tissue most severely affected, pathways implicated in ion transport were observed; effective nutrient transport from mother to offspring plays a critical role in normal offspring growth and development. To further explore the potential impact DNA methylation has on normal biological functions and important developmental pathways, DAVID Pathway Analysis was used, which classifies a large list of genes into functionally related groups of genes to unravel the biological content/similarities (Huang 2009).

The results concluded that in male ART-embryos and placenta, the most significant enriched pathways were related to cell and biological adhesion — when cells interact and attach to a surface, substrate, or another cell, explaining the increased rates of neurodevelopmental abnormalities observed in ART-conceived embryos, as well as genes in the cadherin and claudin families — genes known to play a role in neural tube defects, which are birth defects of the brain, spine, or spinal cord (MedlinePlus).

The picture shows Spina Bifida (MedlinePlus), one of the most common neural tube defects, in which the spinal column doesn’t close completely and nerve damage causes paralysis in the legs.

In ART female placenta, pathways related to normal embryonic development, such as neuron differentiation and signaling, were significantly enriched. In the genes associated with enriched biological pathways, only the ART placenta exhibited significant enrichment in any particular one; most of the enriched pathways were related to neurodevelopmental pathways, including neuron differentiation and development, and axonogenesis — the formation of new axons, which are part of a nerve cell that sends signals to other parts of the body. For example, if you were to put your hand on a hot oven, signals will travel throughout your nervous system to your brain, telling you to remove your hand from the oven; these signals are carried by axons. Together, the results suggest that DNMT-10 deficiency and ART interact to affect pathways involved in poor embryonic outcome.

Additionally, several mouse experiments provide evidence that ART can disrupt normal DNA methylation patterning in embryos (and placenta) which helps reflects the report of human ART with epigenetic defects in offspring. It is difficult to determine how the underlying infertility of individuals seeking ART interacts with the techniques used, and whether the defects are due to infertility or ARTs. The goal of the experiment was to test if alterations in oocyte quality, which match conditions found in older infertile women, make embryos more prone to the consequences of ART. Using a mouse model of females with approximately half of normal DNMT-10 levels in their oocytes, DNMT-10 deficiency and ART techniques interact to make both developmental and epigenetic outcomes in a sex-specific manner worse.

The ART procedures used in the experiment, superovulation — the process of inducing a woman to release more than one egg a month — and embryo transfer (embryos are placed into the uterus of a female with the intent to establish a pregnancy), are similar to those used previously in a study when there was no detected effect on methylation in the absence of DNMT-10 deficiency. This deviation could be due to combined effects of ART and DNMT-10 deficiency or just the DNMT-10 deficiency.

In sum, subfertility, specifically DNMT-10 deficiency combined with ART, appears to have negative impacts on the embryo, including developmental abnormalities and sensitivity in the placenta. While at first glance assisted reproductive technologies seem like a great option, it’s important to understand that even these modern-day advances can have consequences in relation to developmental and cognitive abnormalities. Because the enzyme DNMT-10 is critical to development and ART affects the placenta severely, nutrient transport from mother to embryo may be weak. Knowing the quality of the mother’s eggs first is critical for genetic counselors to communicate the pros and cons to further guide any couples with a high risk of passing on a genetic disorder.

Although researchers are still working to improve this technique, ART has provided people with an amazing option that didn’t exist before. The successful process is a work of ART, and I’m a work of ART.

References:

1. Ding, Karisa. (2016). Fertility Treatment: Assisted reproductive technologies (ART). babycenter. Retrieved from >https://www.babycenter.com/0_fertility-treatment-assisted-reproductive-technologies-art_4093.bc#articlesection2
2. Lee, Y.K., Jin, S., Duan, S., Lim, Y.C., Ng, D.P., Lin, X.M., Yeo, G.S., and Ding, C. (2014). Improved reduced representation bisulfite sequencing for epigenomic profiling of clinical samples. Biol. Proced. Online 16, 1–9222–16–1.
3. Robert J. Brooker, Eric P. Widmaier, Linda E. Graham & Peter D. Stiling. Biology. 4, (Mc-Graw Hill Education, 2017).
4. Tennessee Reproductive Medicine. Superovulation. Retrived from: >https://trmbaby.com/library/treatments/superovulation/
5. Article: Whidden, L. et al. Compromised oocyte quality and assisted reproduction contribute to sex-specific effects on offspring outcomes and epigenetic patterning. Human Molecular Genetics 25, 4649–4660 (2016).
6. Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nature Protoc. 2009;4(1):44–57. Retrieved from: >https://david-d.ncifcrf.gov/
7. Image of Craniofacial Abnormality. Retrieved from: >https://medlineplus.gov/craniofacialabnormalities.html
8. Image of ZIFT. Retrieved from: >http://www.jetanin.com/mirror/plugin/ckeditor/ckfinder/userfiles/images/zift01.jpg
9. https://medlineplus.gov/neuraltubedefects.html
10. Image of Spina Bifida. Retrieved from: >https://upload.wikimedia.org/wikipedia/commons/thumb/7/7e/Spina-bifida.jpg/300px-Spina-bifida.jpg