Faculty Bibliography

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.

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.

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

Importance/UNASSIGNED:It is known that oocytes undergo aging that is caused by exposure to an aged ovarian microenvironment. Telomere length in mouse and bovine oocytes declines with age, and age-associated telomere shortening in oocytes is considered a sign of poor development competency. Women with advanced age undergoing assisted reproductive technologies have poor outcomes because of increasing aneuploidy rates with age. Research has shown that aneuploidy is associated with DNA damage, reactive oxygen species, and telomere dysfunction. Objective/UNASSIGNED:In this review, we focus on the possible relationship between telomere dysfunction and aneuploidy in human early embryo development and several reproductive and perinatal outcomes, discussing the mechanism of aneuploidy caused by telomere shortening and fusion in human embryos. Evidence Acquisition/UNASSIGNED:We reviewed the current literature evidence concerning telomere dysfunction and aneuploidy in early human embryo development. Results/UNASSIGNED:Shorter telomeres in oocytes, leukocytes, and granulosa cells, related to aging in women, were associated with recurrent miscarriage, trisomy 21, ovarian insufficiency, and decreasing chance of in vitro fertilization success. Telomere length and telomerase activity in embryos have been related to the common genomic instability at the cleavage stage of human development. Complications of assisted reproductive technology pregnancies, such as miscarriage, birth defects, preterm births, and intrauterine growth restriction, also might result from telomere shortening as observed in oocytes, polar body, granulosa cells, and embryos. Conclusions and Relevance/UNASSIGNED:Telomere length clearly plays an important role in the development of the embryo and fetus, and the abnormal shortening of telomeres is likely involved in embryo loss during early human development. However, telomere fusion studies have yet to be performed in early human development.

Study question: Do young women with idiopathic early ovarian ageing have changes in telomere length and epigenetic age indicating accelerated biological aging? Summary answer: The telomere length and epigenetic age were comparable to those in young women with normal ovarian ageing. What is known already: Increased risk of several health events usually considered to be age-related such as cardiovascular disease, osteoporosis, over-all morbidity and mortality have been associated with premature and early menopause when compared to the risk in women with normal menopausal age suggesting an accelerated general ageing process associated to early ovarian ageing. It is unclear whether the onset of this process may start before menopause. Study design, size, duration: A prospective cohort study. Young women (<= 37 years) having ART at two Danish Public fertility clinics during the period 2016 to 2018 were divided into two groups dependent on their ovarian reserve status: early ovarian ageing (EOA) (N=55) and normal ovarian ageing (NOA)( N=52). Number of oocytes harvested in first and subsequent cycles was used as a marker of ovarian reserve. Blood samples was drawn at time of oocyte retrieval to assess biological age. Participants/materials, setting, methods: EOA was defined as >= 2 IVF cycles with <= 5 harvested oocytes despite sufficient stimulation with FSH and NOA as >=8 oocytes harvested in minimum 1 cycle. Known causes influencing the ovarian reserve (endometriosis, ovarian surgery, etc.) was reason for exclusion. Relative telomere length (qPCR) and epigenetic age acceleration (DNA methylation levels) were measured in white blood cells as markers of accelerated biological ageing. Main results and the role of chance: Relative telomere length was comparable with a mean of 0.46 (+/- sd 0.12) in the EOA group and 0.47 (0.14) in the normal ovarian ageing group (p=0.64). The difference of predicted mean epigenetic age and mean chronological age (i.e. epigenetic age acceleration) was, insignificantly, 0.5 years older in the EOA group when compared to the NOA group( (-1.02 years (2.62) and -1.57 years (2.56), respectively, p=0.27)), but this difference disappeared when adjusting for chronological age. Limitations, reasons for caution: Discrete changes in epigenetic age acceleration may not have been captured as the study only had power to detect an age acceleration of >= 2 years. Wider implications of the findings: By analysis of biomarkers for ageing in whole blood, we did not find any indications of a premenopausal accelerated aging in young women with idiopathic EOA. Further investigations in a similar cohort of premenopausal women is needed to fully elucidate the potential relationship between premenopausal accelerated biological ageing and EOA

Study question: Do human sperm contain novel LINE-1 insertions and are they affected by paternal age? Summary answer: Human sperm contain novel LINE-1 insertions. Their location or number are not affected by paternal age. What is known already: LINE-1 comprises 17% of the human genome and some LINE-1s are the only autonomous retrotransposons in humans. Retrotransposons influence genomic instability and/or regulation if new retrotransposition events disrupt coding or regulatory regions in the host genome. Demethylation during germ cell development de-represses retrotransposons. Advanced paternal age is associated with genomic instability. Previously we showed that sperm LINE-1 copy number decreases with paternal age. We hypothesize that human sperm exhibit De novo retrotransposition and that sperm from older men contain increased novel LINE-1 insertions. Study design, size, duration: Cross-sectional case-control study with semen samples collected between February to July 2020. Participants/materials, setting, methods: Normospermic sperm samples (n=10; 5 <35 years old and 5 >=45 years old) obtained from consenting men undergoing IVF at NYU Fertility Center were submitted to a novel method, single cell Transposon Insertion Profiling by Sequencing (scTIPseq) to identify and map LINE-1 insertions in human sperm. TIPseqHunter, a custom bioinformatics pipeline, compared the architecture of sperm LINE-1 to known LINE-1 insertions from the European database of human specific LINE-1 (L1Hs) retrotransposon insertions in humans (euL1db). Main results and the role of chance: TIPseq identified 17 novel insertions in sperm, 8 from older (>= 45 years) and 9 in younger men (<35 years). New insertions were mainly intergenic or intronic, including AC007402 (2/10), TMEM163 (2/7), CTTNBP2NL (3/5), AC107023 (3/3), TMC2 (2/19), MacroD2 (2/6), RAB3C (3/4), LINC02664 (1/1), AC079052 (2/3) and AC017091 (4/4). One novel insertion (<35 years old) hits a known regulatory element. Only one sample (>= 45 years old) did not exhibit any new insertion. The location or number of novel insertions did not differ by paternal age. Limitations, reasons for caution: The small sample-size and use of normospermic specimens limit interpretation of paternal age effect on LINE-1. Besides, the novel insertions could be polymorphic sites that have low allele frequency and thus have not yet been described. Wider implications of the findings: This study for the first time reports novel LINE-1 insertions in human sperm, demonstrating that scTIPseq method is a feasible technique, and identifying new contributions to genetic diversity in the human germ line. Further studies are needed to evaluate the impact of these insertions on sperm function