Faculty Bibliography

OBJECTIVE:Millions of babies have been conceived by IVF, yet debate about its safety to offspring continues. We hypothesized that superovulation and in vitro fertilization (IVF) promote genomic changes, including altered telomere length (TL) and activation of the retrotransposon LINE-1 (L1), and tested this hypothesis in a mouse model. MATERIAL AND METHODS/METHODS:Experimental study analyzing TL and L1 copy number in C57BL/6 J mouse blastocysts in vivo produced from natural mating cycles (N), in vivo produced following superovulation (S), or in vitro produced following superovulation (IVF). We also examined the effects of prolonged culture on TL and L1 copy number in the IVF group comparing blastocysts cultured 96 h versus blastocysts cultured 120 h. TL and L1 copy number were measured by Real Time PCR. RESULTS:TL in S (n = 77; Mean: 1.50 ± 1.15; p = 0.0007) and IVF (n = 82; Mean: 1.72 ± 1.44; p < 0.0001) exceeded that in N (n = 16; Mean: 0.61 ± 0.27). TL of blastocysts cultured 120 h (n = 15, Mean: 2.14 ± 1.05) was significantly longer than that of embryos cultured for 96 h (n = 67, Mean: 1.63 ± 1.50; p = 0.0414). L1 copy number of blastocysts cultured for 120 h (n = 15, Mean: 1.71 ± 1.49) exceeded that of embryos cultured for 96 h (n = 67, Mean: 0.95 ± 1.03; p = 0.0162). CONCLUSIONS:Intriguingly ovarian stimulation, alone or followed by IVF, produced embryos with significantly longer telomeres compared to in vivo, natural cycle-produced embryos. The significance of this enriched telomere endowment for the health and longevity of offspring born from IVF merit future studies.

BACKGROUND:Psychological, emotional, and mental distress affects many patients who experience early pregnancy loss (EPL). A common concern is that the patient's actions or choices caused the loss. Understanding the cause of EPL may improve the distress of EPL patients and their partners. Chromosomal abnormalities leading to a significant portion of EPL. Cell-free DNA (cfDNA) testing, a non-invasive test providing high quality information about the chromosomal makeup of a fetus, may offer assurance that a fetal abnormality caused the loss, and provide more certainty or closure in processing EPL. CfDNA may be a useful adjunct to patient-centered care in the setting of EPL. This commentary explores the possibility of cfDNA testing in lessening the emotional distress that often accompanies EPL. METHODS:The peer reviewed literature was explored for manuscripts addressing (1) the potential for cfDNA serum testing for patients experiencing EPL and screening products of conception to determine the cause of EPL; and/or (2) the impact that information might have on the psychological morbidity of EPL for patients and their partners. Themes generated from extracted data were used to generate key questions for future research. RESULTS:Preliminary findings suggest fetal fraction values are instrumental in the success of cfDNA testing, and a successful cfDNA testing experience can have a positive impact on patients. CONCLUSIONS:Ultimately, we conclude cfDNA testing could have a positive impact in patient care and improve the well-being of patients undergoing the emotional toll of EPL by reducing feelings of guilt and providing closure to those who learn the loss was associated with chromosomal abnormality. Further trials and studies that explore the intersection of mental health of EPL on patients should explore the efficacy of cfDNA testing as an adjunct to patient-centered care in these cases.

Objective/UNASSIGNED:To describe a unique case of primary ovarian insufficiency and review the systemic barriers in place that hinder reproductive autonomy for Black women who require third-party reproduction. Design/UNASSIGNED:Case report and review of the literature. Setting/UNASSIGNED:Safety-net hospital in an urban community. Patients/UNASSIGNED:A 36-year-old Black woman, gravida 0, with primary ovarian insufficiency who desires future fertility but is restricted by systemic barriers. Interventions/UNASSIGNED:Chromosome analysis. Main Outcome Measures/UNASSIGNED:Not applicable. Results/UNASSIGNED:Balanced reciprocal translocation between chromosomes 1 and 13: 46,XX,t(1;13)(q25;q14.1). Conclusions/UNASSIGNED:The field of assisted reproductive technology has evolved at an exponential rate, yet it unfortunately benefits some and not all. It is imperative that when we advocate for full spectrum infertility care, that this encompasses everyone. As we continue to further study and develop assisted reproductive technology, we must not forget to consider the factors leading to racial and socioeconomic disparities in reproductive care access, utilization, and outcomes.

Maintenance of genome integrity in the germline and in preimplantation embryos is crucial for mammalian development. Epigenetic remodeling during primordial germ cell (PGC) and preimplantation embryo development may contribute to genomic instability in these cells, since DNA methylation is an important mechanism to silence retrotransposons. Long interspersed elements 1 (LINE-1 or L1) are the most common autonomous retrotransposons in mammals, corresponding to approximately 17% of the human genome. Retrotransposition events are more frequent in germ cells and in early stages of embryo development compared with somatic cells. It has been shown that L1 activation and expression occurs in germline and is essential for preimplantation development. In this review, we focus on the role of L1 retrotransposon in mouse and human germline and early embryo development and discuss the possible relationship between L1 expression and genomic instability during these stages. Although several studies have addressed L1 expression at different stages of development, the developmental consequences of this expression remain poorly understood. Future research is still needed to highlight the relationship between L1 retrotransposition events and genomic instability during germline and early embryo development.

Proper telomere length is essential for indefinite self-renewal of embryonic stem (ES) cells and cancer cells. Telomerase-deficient late generation mouse ES cells and human ALT cancer cells are able to propagate for numerous passages, suggesting telomerase-independent mechanisms responding for telomere maintenance. However, the underlying mechanisms ensuring the telomere length maintenance are unclear. Here, using late generation telomerase KO (G4 Terc^-/-) ESCs as a model, we show that Zscan4, highly upregulated in G4 Terc^-/- ESCs, is responsible for the prolonged culture of these cells with stably short telomeres. Mechanistically, G4 Terc^-/- ESCs showed reduced levels of DNA methylation and H3K9me3 at Zscan4 promoter and subtelomeres, which relieved the expression of Zscan4. Similarly, human ZSCAN4 was also derepressed by reduced H3K9me3 at its promoter in ALT U2 OS cells, and depletion of ZSCAN4 significantly shortened telomeres. Our results define a similar conserved pathway contributing to the telomere maintenance in telomerase-deficient late generation mESCs and human ALT U2OS cancer cells.

Parthenogenetic embryos, created by activation and diploidization of oocytes, arrest at mid-gestation for defective paternal imprints, which impair placental development. Also, viable offspring has not been obtained without genetic manipulation from parthenogenetic embryonic stem cells (pESCs) derived from parthenogenetic embryos, presumably attributable to their aberrant imprinting. We show that an unlimited number of oocytes can be derived from pESCs and produce healthy offspring. Moreover, normal expression of imprinted genes is found in the germ cells and the mice. pESCs exhibited imprinting consistent with exclusively maternal lineage, and higher X-chromosome activation compared to female ESCs derived from the same mouse genetic background. pESCs differentiated into primordial germ cell-like cells (PGCLCs) and formed oocytes following in vivo transplantation into kidney capsule that produced fertile pups and reconstituted ovarian endocrine function. The transcriptome and methylation of imprinted and X-linked genes in pESC-PGCLCs closely resembled those of in vivo produced PGCs, consistent with efficient reprogramming of methylation and genomic imprinting. These results demonstrate that amplification of germ cells through parthenogenesis faithfully maintains maternal imprinting, offering a promising route for deriving functional oocytes and having potential in rebuilding ovarian endocrine function.

PURPOSE/OBJECTIVE:To investigate whether inhibition of LINE-1 affects telomere reprogramming during 2-cell embryo development. METHODS:Mouse zygotes were cultured with or without 1 µM azidothymidine (AZT) for up to 15 h (early 2-cell, G1/S) or 24 h (late 2-cell, S/G2). Gene expression and DNA copy number were determined by RT-qPCR and qPCR respectively. Immunostaining and telomeric PNA-FISH were performed for co-localization between telomeres and ZSCAN4 or LINE-1-Orf1p. RESULTS:LINE-1 copy number was remarkably reduced in later 2-cell embryos by exposure to 1 µM AZT, and telomere lengths in late 2-cell embryos with AZT were significantly shorter compared to control embryos (P = 0.0002). Additionally, in the absence of LINE-1 inhibition, Dux, Zscan4, and LINE-1 were highly transcribed in early 2-cell embryos, as compared to late 2-cell embryos (P < 0.0001), suggesting that these 2-cell genes are activated at the early 2-cell stage. However, in early 2-cell embryos with AZT treatment, mRNA levels of Dux, Zscan4, and LINE-1 were significantly decreased. Furthermore, both Zscan4 and LINE-1 encoded proteins localized to telomere regions in 2-cell embryos, but this co-localization was dramatically reduced after AZT treatment (P < 0.001). CONCLUSIONS:Upon inhibition of LINE-1 retrotransposition in mouse 2-cell embryos, Dux, Zscan4, and LINE-1 were significantly downregulated, and telomere elongation was blocked. ZSCAN4 foci and their co-localization with telomeres were also significantly decreased, indicating that ZSCAN4 is an essential component of the telomere reprogramming that occurs in mice at the 2-cell stage. Our findings also suggest that LINE-1 may directly contribute to telomere reprogramming in addition to regulating gene expression.

PURPOSE/OBJECTIVE:Millions of pregnant, HIV-infected women take reverse transcriptase inhibitors, such as zidovudine (azidothymidine or AZT), during pregnancy. Reverse transcription plays important roles in early development, including regulation of telomere length (TL) and activity of transposable elements (TE). So we evaluated the effects of AZT on embryo development, TL, and copy number of an active TE, Long Interspersed Nuclear Element 1 (LINE-1), during early development in a murine model. DESIGN/METHODS:Experimental study. METHODS:In vivo fertilized mouse zygotes from B6C3F1/B6D2F1 mice were cultured for 48 h in KSOM with no AZT (n = 45), AZT 1 μM (n = 46) or AZT 10 μM (n = 48). TL was measured by single-cell quantitative PCR (SC-pqPCR) and LINE-1 copy number by qPCR. The percentage of morulas at 48 h, TL and LINE-1 copy number were compared among groups. RESULTS:Exposure to AZT 1 μM or 10 μM significantly impairs early embryo development. TL elongates from oocyte to control embryos. TL in AZT 1 μM embryos is shorter than in control embryos. LINE-1 copy number is significantly lower in oocytes than control embryos. AZT 1 μM increases LINE-1 copy number compared to oocytes controls, and AZT 10 μM embryos. CONCLUSION/CONCLUSIONS:AZT at concentrations approaching those used to prevent perinatal HIV transmission compromises mouse embryo development, prevents telomere elongation and increases LINE-1 copy number after 48 h treatment. The impact of these effects on the trajectory of aging of children exposed to AZT early during development deserves further investigation.