Young women with poor ovarian response exhibit epigenetic age acceleration based on evaluation of white blood cells using a DNA methylation-derived age prediction model.

STUDY QUESTION: Is poor ovarian response associated with a change in predicted age based on a DNA methylation-derived age prediction model (the Horvath algorithm) in white blood cells (WBCs) or cumulus cells (CCs)? SUMMARY ANSWER: In young women, poor ovarian response is associated with epigenetic age acceleration within WBC samples but is not associated with age-related changes in CC. WHAT IS KNOWN ALREADY: The majority of human tissues follow predictable patterns of methylation which can be assessed throughout a person's lifetime. DNA methylation patterns may serve as informative biomarkers of aging within various tissues. Horvath's 'epigenetic clock', which is a DNA methylation-derived age prediction model, accurately predicts a subject's true chronologic age when applied to WBC but not to CC. STUDY DESIGN, SIZE, DURATION: A prospective cohort study was carried out involving 175 women undergoing ovarian stimulation between February 2017 and December 2018. Women were grouped according to a poor (≤5 oocytes retrieved) or good (>5 oocytes) response to ovarian stimulation. Those with polycystic ovary syndrome (PCOS) (n = 35) were placed in the good responder group. PARTICIPANTS/MATERIALS, SETTING, METHODS: DNA methylation patterns from WBC and CC were assessed for infertile patients undergoing ovarian stimulation at a university-affiliated private practice. DNA was isolated from peripheral blood samples and CC. Bisulfite conversion was then performed and a DNA methylation array was utilized to measure DNA methylation levels throughout the genome. Likelihood ratio tests were utilized to assess the relationship between predicted age, chronologic age and ovarian response. MAIN RESULTS AND THE ROLE OF CHANCE: The Horvath-predicted age for WBC samples was consistent with patients' chronologic age. However, predicted age from analysis of CC was younger than chronologic age. In subgroup analysis of women less than 38 years of age, poor ovarian response was associated with an accelerated predicted age in WBC (P = 0.017). Poor ovarian response did not affect the Horvath-predicted age based on CC samples (P = 0.502). No alternative methylation-based calculation was identified to be predictive of age for CC. LIMITATIONS, REASONS FOR CAUTION: To date, analyses of CC have failed to identify epigenetic changes that are predictive of the aging process within the ovary. Despite the poor predictive nature of both the Horvath model and the novel methylation-based age prediction model described here, it is possible that our efforts failed to identify appropriate sites which would result in a successful age-prediction model derived from the CC epigenome. Additionally, lower DNA input for CC samples compared to WBC samples was a methodological limitation. We acknowledge that a universally accepted definition of poor ovarian response is lacking. Furthermore, women with PCOS were included and therefore the group of good responders in the current study may not represent a population with entirely normal methylation profiles. WIDER IMPLICATIONS OF THE FINDINGS: The process of ovarian and CC aging continues to be poorly understood. Women who demonstrate poor ovarian response to stimulation represent a common clinical challenge, so clarifying the exact biological changes that occur within the ovary over time is a worthwhile endeavor. The data from CC support a view that hormonally responsive tissues may possess distinct epigenetic aging patterns when compared with other tissue types. Future studies may be able to determine whether alternative DNA methylation sites can accurately predict chronologic age or ovarian response to stimulation from CC samples. Going forward, associations between epigenetic age acceleration and reproductive and general health consequences must also be clearly defined. STUDY FUNDING/COMPETING INTEREST(S): No external funding was obtained for the study and there are no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Three-dimensional ultrasound diagnosis of T-shaped uterus is associated with adverse pregnancy outcomes after embryo transfer.

RESEARCH QUESTION: Is T-shaped uterine cavity morphology associated with adverse pregnancy outcomes after transfer of a single thawed euploid blastocyst? DESIGN: In this secondary analysis of a prospective cohort study, 648 patients with three-dimensional ultrasound (3D-US) data obtained on the day before embryo transfer were categorized into three groups according to uterine cavity morphology: normal (n = 472), intermediate (n = 166) and T-shaped (n = 10). Quantitative uterine cavity dimensions were used to evaluate uterine cavity morphology. Pregnancy outcomes, including live birth, clinical miscarriage and ectopic pregnancy, were compared among the groups. RESULTS: The prevalence of a T-shaped uterus in this cohort was 1.5%. Uterine cavity morphology was strongly associated with the ratio of interostial distance and isthmic diameter (P < 0.01). Live birth rates were 66.5% for normal, 65.7% for intermediate and 40.0% for T-shaped cavity morphology. Women with a T-shaped uterus had an increased risk of clinical miscarriage (40.0% versus 7.0% for normal and 9.0% for intermediate cavity morphology, P < 0.01) and ectopic pregnancy (10.0% versus 1.1% for normal and 1.9% for intermediate cavity morphology, P = 0.05). When evaluating interostial distance and isthmic diameter ratio to determine pregnancy outcomes, a cut-off value of 2 was noted to have weak predictive value for live birth, but not clinical miscarriage or ectopic pregnancy. CONCLUSIONS: T-shaped uterine cavity morphology is associated with adverse pregnancy outcomes after transfer of a single thawed euploid blastocyst. Given the low prevalence of this condition, quantifying the magnitude of risk will require a larger cohort of patients.

Relation between plasma phospholipid oleic acid and risk of heart failure.

BACKGROUND AND AIMS: Although the association between monounsaturated fatty acids (MUFA) and risk factors for heart failure (HF) has been reported, it is unclear whether oleic acid, the predominant MUFA in olive oil, plays a role in the development of HF. Consequently, we sought to examine the relation of plasma phospholipid oleic acid with HF in a male cohort. In a secondary analysis, we examined the relation of the ratio of plasma monounsaturated-to-saturated fatty acids (MUFA: SFA) with HF. METHODS: This prospective nested case-control study was based on 788 incident HF cases and 788 controls from the Physicians' Health Study. Plasma phospholipid fatty acids were measured using gas chromatography and incident HF was self-reported via annual follow-up questionnaires and validated in a subsample using medical records. RESULTS: The mean age was 58.7 years at blood collection. In a conditional logistic regression, multivariable adjusted-odds ratios (95% confidence interval) for HF across consecutive quartiles of oleic acid were 1.0 (reference), 1.10 (0.79-1.54), 1.02 (0.72-1.44), and 1.05 (0.72-1.54). For MUFA:SFA ratio, corresponding odds ratios (95% CI) for HF were 1.0 (ref), 1.12 (0.80-1.58), 1.19 (0.84-1.68), and 0.97 (0.66-1.42). CONCLUSIONS: Our data do not lend support to an association between plasma phospholipid oleic acid or MUFA: SFA ratio and the risk of HF. These results warrant confirmation in the general population including women and other ethnic groups.

The dilemma of aneuploidy screening on low responders.

PURPOSE OF REVIEW: Preimplantation genetic testing for aneuploidy (PGT-A) has been demonstrated to improve implantation and pregnancy rates and decrease miscarriage rates over standard morphology-based embryo selection. However, there are limited data on its efficacy in patients with diminished ovarian reserve or a poor response to stimulation who may have fewer embryos to select amongst. RECENT FINDINGS: Early findings demonstrate that PGT-A reduces the miscarriage rate and decreases the time to delivery in poor responders. These studies highlight the importance of designing trials that compare outcomes over multiple cycles as the benefit of PGT-A in this patient population lies in eliminating the time lost to futile transfers of aneuploid embryos. Furthermore, recent studies have demonstrated that a catch-all category of 'poor responder' may need to be reevaluated as different subpopulations of patients with low response exhibit different clinical characteristics. SUMMARY: More information is needed on characterizing the physiology of ovarian aging across multiple phenotypes of diminished ovarian reserve and establishing the predictive value of aneuploid results across multiple PGT-A platforms. However, initial data suggests benefit of PGT-A in poor responders.

Levels of trophectoderm mitochondrial DNA do not predict the reproductive potential of sibling embryos.

Study question: What is the predictive value of trophectoderm mitochondrial DNA (mtDNA) quantity for blastocyst reproductive potential? Summary answer: This study demonstrates that, within a given cohort, mtDNA quantitation does not distinguish between embryos that implant and embryos that do not implant after double embryo transfer (DET). What is already known: An association between implantation failure and increased quantities of mtDNA has been observed in two studies but not in a third. Study design, size and duration: A total of 187 patients (nine who received donor oocytes) with DET of one male and one female euploid blastocyst were included in this retrospective study, with 69 singleton deliveries providing the primary dataset to evaluate the predictive value of mtDNA for reproductive potential between January 2010 and July 2016. Participants/materials, setting and method: MtDNA was quantified in cell lines to validate the quantitative PCR assay on limited quantities of starting material and then applied to 374 blastocyst biopsies. Pregnancies resulting in a singleton outcome were analyzed and newborn gender was utilized as a means to identify the implanted embryo. MtDNA quantity was then compared between implanted and non-implanted embryos in order to define the predictive value of mtDNA content for reproductive potential in this subset of patients. Main results and the role of chance: An initial comparison of mtDNA levels between all successful and unsuccessful embryos revealed no significant differences. In order to control for patient-specific variables, gender was subsequently used to identify the implanted embryo in DETs resulting in a singleton (n = 69). No systematic difference in relative mtDNA quantity was detected between implanted and non-implanted embryos. Limitations, reasons for caution: This study was conducted at a single center and did not evaluate the entire cohort of embryos from each patient to evaluate cohort specific variation in mtDNA quantity. Although the largest of its kind so far, the sample size of DETs leading to a singleton was relatively small. Wider implications of the findings: These data highlight the importance of control over patient-specific variables when evaluating candidate biomarkers of reproductive potential. All current available data suggest that mtDNA quantification needs further study before its clinical use to augment embryo selection. Study funding/competing interests: The authors have no potential conflict of interest to declare. No external funding was obtained for this study. Trial registration number: Not applicable.

Oxygen tension in embryo culture: does a shift to 2% O2 in extended culture represent the most physiologic system?

There has been much debate regarding the optimal oxygen tension in clinical embryo culture. The majority of the literature to date has compared 5% oxygen to atmospheric levels (20-21%). While the majority of modern IVF labs have accepted the superiority of 5% oxygen tension, a new debate has emerged regarding whether a further reduction after day 3 of development represents the most physiologic system. This new avenue of research is based on the premise that oxygen tension is in fact lower in the uterus than in the oviduct and that the embryo crosses the uterotubal junction sometime on day 3. While data are currently limited, recent experience with ultra-low oxygen (2%) after day 3 of development suggests that the optimal oxygen tension in embryo culture may depend on the stage of development. This review article will consider the current state of the literature and discuss ongoing efforts at studying ultra-low oxygen tension in extended culture.

FMR1 gene CGG repeat variation within the normal range is not predictive of ovarian response in IVF cycles.

The relationship between FMR1 CGG premutation status and decreased ovarian responsiveness is well established. The association between FMR1 CGG repeat number in the currently defined normal range (less than 45 repeats) and ovarian reserve, however, is controversial. This retrospective study examined whether variation in CGG repeat number in the normal range was associated with markers of ovarian response in IVF cycles. The first IVF cycle of 3006 patients with FMR1 CGG repeat analysis was examined. Only patients carrying two alleles with less than 45 CGG repeats were included for analysis. The CGG repeat number furthest from the modal peak was plotted against number of mature oocytes retrieved and no correlation was identified. Patients were also separated into biallelic genotype groups, based on the recently proposed narrower "new normal" range of 26-34 CGG repeats. A linear regression showed that none of the biallelic genotype groups were associated with a decreased oocyte yield. The euploidy rates after comprehensive chromosomal screening were equivalent among the genotype groups. No difference was found in the rate of cycle cancellation for poor response. Despite increasing use, FMR1 CGG repeats in the normal range cannot be used as a predictor of ovarian response to gonadotrophin stimulation.

Deliveries from trophectoderm biopsied, fresh and vitrified blastocysts derived from polar body biopsied, vitrified oocytes.

This longitudinal study reports preliminary findings of six patients who underwent first polar body biopsy followed by oocyte vitrification. All oocytes were warmed, inseminated by intracytoplasmic sperm injection and cultured to blastocyst. All suitable blastocysts underwent trophectoderm biopsy for aneuploidy screening, and supernumerary blastocysts were vitrified. Euploid blastocysts were transferred either fresh or in a subsequent programmed cycle. Of the 91 metaphase II oocytes, 30 had euploid first polar bodies. Development to blastocyst was more likely in oocytes with a euploid first polar body (66.7% versus 24.6%; P < 0.001). Nineteen euploid blastocysts were produced: 10 from oocytes with a euploid first polar body and nine from oocytes with an aneuploid first polar body. Five out of six patients (83%) had a live birth or ongoing pregnancy at the time of analysis. Eleven euploid blastocysts have been transferred and seven implanted (64%). Although the chromosomal status of the first polar body was poorly predictive of embryonic ploidy, an association was found between chromosomal status of the first polar body and development to blastocyst. Further study is required to characterize these relationships, but proof of concept is provided that twice biopsied, twice cryopreserved oocytes and embryos can lead to viable pregnancies.

Identification and expression of GnRH2 and GnRH3 in the black sea bass (Centropristis striata), a hermaphroditic teleost.

We cloned two cDNAs for two gonadotropin-releasing hormones, GnRH2 (chicken GnRH-II) and GnRH3 (salmon GnRH), respectively, from the black sea bass (Centropristis striata). Black sea bass are protogynous hermaphroditic teleosts that change from females to males between 2 and 5 years of age. Similar to other GnRH precursors, the precursors of black sea bass GnRH2 and GnRH23 consisted of a signal peptide, decapeptide, a downstream processing site, and a GnRH-associated peptide. Our analyses failed to identify GnRH1. GnRH3 precursor transcript was more widely distributed in a variety of tissues compared with GnRH2. Further examination of GnRH expression and gonadal histology was done in black sea bass from three different size groups: small (11.4-44.1 g), medium (179.4-352.2 g) and large (393.8-607.3 g). Interestingly, GnRH3 expression occurred only in the pituitaries of males in the small and medium groups compared with expression of GnRH2. Future functional studies of the sea bass GnRHs will be valuable in elucidating the potential underlying neuroendocrine mechanisms of black sea bass reproduction and may ultimately contribute to management advances in this commercially important fish.