Faculty Bibliography

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.

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.

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 evaluate whether young women with idiopathic early ovarian aging, as defined by producing fewer oocytes than expected for a given age over multiple in vitro fertilization (IVF) cycles, have changes in telomere length and epigenetic age indicating accelerated biological aging (i.e., increased risk of morbidity and mortality). METHODS:A prospective cohort study was conducted at two Danish public fertility clinics. A total of 55 young women (≤ 37 years) with at least two IVF cycles with ≤ 5 harvested oocytes despite sufficient stimulation with follicle-stimulating hormone (FSH) were included in the early ovarian aging group. As controls, 52 young women (≤ 37 years) with normal ovarian function, defined by at least eight harvested oocytes, were included. Relative telomere length (rTL) and epigenetic age acceleration (AgeAccel) were measured in white blood cells as markers of premenopausal accelerated biological aging. RESULTS:rTL was comparable with a mean of 0.46 (± SD 0.12) in the early ovarian aging group and 0.47 (0.14) in the normal ovarian aging group. The AgeAccel of the early ovarian aging group was, insignificantly, 0.5 years older, but this difference disappeared when adjusting for chronological age. Sub-analysis using Anti-Müllerian hormone (AMH) as selection criterion for the two groups did not change the results. CONCLUSION/CONCLUSIONS:We did not find any indications of accelerated aging in whole blood from young women with idiopathic early ovarian aging. Further investigations in a similar cohort of premenopausal women or other tissues are needed to fully elucidate the potential relationship between premenopausal accelerated biological aging and early ovarian aging.

OBJECTIVE: Over 15 million 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. MATERIALS AND METHODS: Experimental laboratory study analyzing C57BL/6J mice produced blastocysts in vivo from natural mating cycles (N), in vivo following superovulation (S), or in vitro following superovulation (IVF). We also examined the effects of prolonged culture on TL and L1 in the IVF group. TL and L1 copy number were measured by Real Time PCR. Following log transformation, analysis of variance with Tukey post-test compared TL and L1 among the 3 groups. Students t test compared TL and L1 between embryos cultured for 120 vs. 96 hrs in the IVF group. Pvalue <0.05 was considered significant. Analyses were performed with SAS 9.4.
RESULT(S): In the IVF group, 10 replicates produced a fertilization rate of 90.52% (95% CI: 85.19-95.85), D4 blastocyst formation rate of 61.90% (95% CI: 52.62-71.19) and cumulative blastocyst rate (D4 plus D5) of 76.19% (CI: 68.04-84.34). 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). L1 copy number in N (n=16; Mean: 0.80+/- 0.31) did not differ from S (n=77; Mean: 1.23+/- 0.75; p=0.1386) or IVF (n=82; Mean: 1.09+/- 1.16; p=0.6709). L1 copy number of embryos from S also did not differ significantly from IVF (n=82; Mean: 1.09+/- 1.16; p=0.0670). TL of blastocysts cultured 120h (n=14, Mean: 2.14 +/- 1.05) was significantly longer than that of embryos cultured for 96h (n=67, Mean: 1.63+/- 1.50, p=0.0414). L1 copy number of blastocysts cultured for 120h (n=15, Mean:1.71+/- 1.49) exceeded that of embryos cultured for 96h (n=67, Mean: 0.95+/- 1.03 p=0.0162).
CONCLUSION(S): Intriguingly ovarian hyperstimulation and 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. The mechanism driving telomere lengthening in response to ovarian stimulation and IVF during early embryo development remains unclear, though may involve activation of L1. Recently we demonstrated a role for L1 in telomere elongation in preimplantation embryos, and Barbara McClintock's Nobel Prizing winning research previously identified activation of retrotransposons as a response to stress. Stress from IVF may elongate telomeres by activating L1. IMPACT STATEMENT: Millions of babies have been born following IVF, yet debate continues about its safety to offspring. We found genomic effects of IVF and ovarian stimulation in mice - telomere elongation and retrotransposon activation. Future studies should examine longevity and/or cancer risk in IVF offspring

OBJECTIVE: To compare telomere length (TL) and telomerase gene expression in human euploid and aneuploid blastocysts generated from IVF treatment. MATERIALS AND METHODS: TL and telomerase gene expression were measured in cryopreserved aneuploid (N=115) and euploid (N=4) human blastocysts donated by 26 patients who consented research under approval of IRB study #16-00154. Blastocysts were classified according to number of aneuploid chromosomes (A1-one segmental error, A2-one whole chromosome error, A3-two chromosomal errors and A4- >= 3 chromosomal errors). Genomic DNA and messenger RNA were separated simultaneously from individual blastocysts after thawing in vitrification-warming media. Telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) mRNA levels were determined by RT-qPCR with GAPDH as internal control, and TL was measured by qPCR with 5s rDNA as internal control. Relative gene expression and TL were calculated by DELTADELTA^Ct method, and GraphPad Prism 8 software was used for statistical analysis.
RESULT(S): TL and telomerase gene expression were not normally distributed, so nonparametric tests were used to compare the medians among groups (Table 1). Median TL, TERTand TERC levels didn't differ by number of chromosome errors nor between aneuploid and euploid groups. Intriguingly, TL, TERT and TERC levels in aneuploid blastocysts tended to be greater compared to euploid blastocysts. TL in blastocysts correlated with telomerase TERT expression (R² =0.054, P = 0.011), but not TERC expression (R² =0.0002, P = 0.865).
CONCLUSION(S): To our knowledge, this is the largest study to measure telomere length and telomerase gene expression in human blastocysts. Our data indicated that telomeres are lengthened and telomerase is activated in aneuploid embryos at blastocyst stage. Moreover, telomere length and telomerase gene TERT in human blastocysts correlate regardless of ploidy status. Like cancer cells, TERT is highly expressed in aneuploid blastocysts. IMPACT STATEMENT: Robust TERT expression and telomere maintenance in aneuploid human blastocysts may explain why extended in vitro culture alone is insufficient to cull out aneuploidy embryos during IVF (Table Presented)

OBJECTIVE: It is well known that the embryo aneuploidy rate increases with women's age [1], but the effect of age on complex chromosomal abnormality (CCA) is less clear. Here, we addressed the relationship between maternal age and CCA with a retrospective cohort study. MATERIALS AND METHODS: We reviewed results of preimplantation genetic testing (PGT) by aCGH or NGS of embryo biopsies performed in an academic IVF unit between 2010 and 2019. We excluded PGT results from single gene disorder and egg donation cycles. CCA was defined as>=3 chromosome abnormalities (whole, partial and/or mosaic). Maternal age was categorized according to SART age groups: <35, 35-37, 38-40, 41-42, and >42 years. Statistical analyses were conducted using GraphPad Prism 8.
RESULT(S): 27,423 embryos were biopsied from 3,501 women aged 23 to 48 years. 4,740 embryos (16%) has CCA. Consistent with prior study [2], the most frequent chromosomes involved in CCA were 22, 16, 21 and 15, with incidences of 30.6%, 29.1%, 26.1% and 25.8% respectively. The number of chromosomal errors (from 3 to 42) involved in CCA did not correlate with maternal age (Spearman r = -0.0149, P = 0.3352). However, the rate of complex abnormal embryos tended to increase with advancing maternal age (9.7%, 11.2%, 10.9%, 24.8% and 43.6% in women aged < 35, 35-37, 38-40, 41-42, and > 42 years, respectively). Women over 40 years old had significantly higher rates of CCA compared to those under 40 years (Chisquare test, P < 0.0001). Surprisingly, the relationship between maternal age and CCA followed a U-shaped curve, decreasing from the 25 to 30 year old group (Pearson r = -0.831, P = 0.04) to the 30 to 35 year old group (Pearson r = 0.093, P = 0.861), then increased markedly in the 35 to 48 year old group (Pearson r = 0.921, P < 0.0001).
CONCLUSION(S):We found that CCA embryos share common features of aneuploidy, such as association with maternal age and preferential involvement of shorter chromosomes i.e. 22, 16, 21 and 15. Unexpectedly, our data showed that the relationship between CCA and maternal age assumes a U shape with increased rates at very young and very old ages. Both meiotic and mitotic errors contribute to chromosomal abnormality, and the contribution of each to CCA merits further investigation. IMPACT STATEMENT: The complex relationship between maternal age and embryo aneuploidy, which approximates a U-shape, may inform optimal timing of elective oocyte freezing and oocyte donation

OBJECTIVE: The causes of spontaneous abortion (SAB) following euploid embryo transfer (EET) remain poorly understood. Here we describe the frequency of aneuploidy in products of conception (POC) and endometrial dysfunction in women who miscarried after EET. MATERIALS AND METHODS: Between 1/2018 - 8/2020, 255 dilation and curettage (D&C) procedures were performed at a large academic IVF center for SAB following EET. Retrospective chart review was performed to identify D&Cs followed with genetic analysis of POCs. Information collected from the medical record included assessments of endometrial dysfunction based on Endometrial Receptivity Assay (ERA), CD138 for chronic endometritis (CE), and/or BCL6 for endometriosis. Exclusion criteria included an abnormal endometrial cavity on imaging. Demographic factors, clinical parameters and IVF/FET outcomes were reviewed. Additionally, retrospective chart review was performed of all ERAs completed at our institution from 12/2018-9/2020.
RESULT(S): Genetic analysis of 67 POCs after D&C following EET were identified. Fifty-nine POCs (88%) were euploid by SNP microarray. Eight (12%) of the POCs displayed genetic abnormalities: 3 trisomies, 2 partial duplications, 2 mosaic trisomies and 1 triploidy of paternal origin. Of the 51 patients who had endometrial biopsy (EMB), 28 (55%) had normal results. Twenty-three (45%) had abnormal results: 18 with CE, 2 with elevated BCL6 and 3 with pre-receptive ERA. The proportion of SABs unexplained by endometrial dysfunction or genetically abnormal POCs was 38% (26). A total of 44 patients underwent repeat EET. Eleven live births (LB) occurred, six after correction of endometrial dysfunction. Eight patients currently have ongoing pregnancy, 2 after treatment for CE. Three patients experienced repeat SAB, 1 following correction of pre-receptive ERA, and 1 after CE treatment. Four patients had implantation failure, 3 following normal EMB and 1 after treatment of CE. Two patients conceived spontaneously and delivered, 1 after treatment for CE, the other after a normal EMB. Upon review of all ERAs, 82 single EET following ERA guidance were identified. Fifty-nine percent (n=48) resulted in ongoing pregnancy or LB. There was no significant difference in ERA result or post ERA transfer outcome based on ethnicity (p= 0.7, p=0.4) or BMI (p= 0.8, 0.9), respectively. There was also no difference in post ERA transfer outcome based on blastocyst age (day 5 or 6) (p=0.5)
CONCLUSION(S): Aneuploidy and/or endometrial factor can contribute to SAB following EET. Aneuploid POCs could have arisen de novo and/or have passed undetected by trophectoderm biopsy and NGS. Our results are consistent with the 1-2% false negative rate reported for PGT-A. Further studies are needed to characterize the sub-chromosomal genetic variations associated with euploid embryo SABs, as well as endometrial function testing. IMPACT STATEMENT: The etiology behind failed EET may involve more discrete entities such as sub-chromosomal abnormalities in addition to aneuploidy and endometrial dysfunction

PURPOSE/OBJECTIVE:Whether differences in stimulation parameters alter the number and proportion of MII oocytes retrieved. METHODS:Records of 2546 patients were examined, looking at age, day 2/3 follicle-stimulating hormone (FSH) and estradiol (E2) levels, total dose of gonadotropins administered (including FSH and human menopausal gonadotropin [hMG]), fraction of hMG administered, number of days of treatment with gonadotropins, and the dose of gonadotropins administered per day. We segregated the patients into 3 different classes depending on the trigger method used and 2 groups based on egg freeze vs. ICSI. Multiple regression methods were used to examine associations between stimulation parameters and the total number of eggs, number of immature oocytes (Poisson regression), and the fraction of retrieved oocytes that were immature (Logistic regression). RESULTS:After adjustments for different triggers and egg freeze versus ICSI, both the #immature oocytes and the immature fraction of oocytes were associated with the total gonadotropin dose (inversely) and the gonadotropin dose/day (positively). Other parameters were associated with the number of immature oocytes but were also associated with the number of oocytes retrieved. CONCLUSIONS:Stimulations using less total gonadotropin and more gonadotropin per day were associated with more immaturity. The type of trigger method used for final maturation was associated with immaturity but was believed to be predominantly due to trigger assignment to patients based on response. The association between use of ICSI and less immaturity was believed to be due to additional time for maturation in the ICSI group.