Exposure to corticosteroids in the first trimester is associated with an increased risk of urogenital congenital anomalies.

STUDY QUESTION: Does maternal exposure to first trimester corticosteroids in IVF/ICSI treatment result in an increased risk of congenital anomalies? SUMMARY ANSWER: Children born with the aid of IVF/ICSI whose mothers were treated with adjuvant corticosteroids during the first trimester had an increased risk of cryptorchidism, hypospadias and talipes. WHAT IS KNOWN ALREADY: Maternal exposure to corticosteroids may increase the risk of congenital anomalies such as cleft palate and neural tube defects. However, the existing studies have conflicting outcomes, are underpowered, and do not study a population undergoing IVF/ICSI, a group known to be at increased risk of abnormalities. STUDY DESIGN, SIZE, DURATION: This retrospective cohort analysis covering Monash IVF fertility clinics in Melbourne, Australia assessed the outcomes of 12 426 live births from both fresh and frozen embryo transfers between 2010 and 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: There were 618 live births included in our study group of mothers exposed to corticosteroids (oral prednisolone or dexamethasone) during their IVF/ICSI treatment, with the remainder of births not exposed to steroids (control, n = 11 808). The primary outcome measured was the presence of congenital anomalies and secondary outcomes were birth weight and gestation length. Multivariate binary logistic regression was used to assess the independent effects of corticosteroid exposure and the freezing of embryos, with adjustment for maternal age at oocyte retrieval, smoking status, number of cycles taken, BMI, etiology of the infertility and the use of ICSI. Results are presented as incidence rate ratios (IRRs) with 95% CIs. MAIN RESULTS AND THE ROLE OF CHANCE: Amongst 12 426 live births, and 597 birth defects, multivariate logistic regression demonstrated there was an increased incidence in talipes equinovarus (1.33% vs 0.32%, adjusted IRR = 4.30, 95% CI = 1.93, 9.58; P < 0.001), hypospadias (0.66% vs 0.18%, adjusted IRR = 5.90, 95% CI = 2.09, 16.69; P = 0.001) and cryptorchidism (0.83% vs 0.19%, adjusted IRR = 5.53, 95% CI = 1.91, 15.42; P = 0.001) in the offspring of mothers exposed to corticosteroids compared to those who were unexposed. The incidence of neither neural tube defects nor cleft palate were significantly increased in babies exposed to corticosteroids. The sex ratio of infants exposed to corticosteroids during a fresh embryo transfer cycle significantly favored males but reverted to the normal sex ratio in infants conceived in frozen embryo transfer cycles. LIMITATIONS, REASONS FOR CAUTION: This was a retrospective observational cohort study using administrative datasets with the potential for measurement error and unobserved confounding. Missing outcome data were obtained from patients using self-report leading to possible ascertainment bias. Given the rare incidence of some of the anomalies assessed, the study was underpowered to identify differences in abnormality rates for some specific anomalies. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study, the largest of its kind, suggest that caution should be heeded when prescribing corticosteroids to women undergoing IVF/ICSI, given that this study has now identified three previously unassociated serious neonatal complications (talipes, hypospadias and cryptorchidism), plus a potential alteration in sex ratio. Physicians should be careful in using corticosteroids in the critical first trimester and should counsel patients regarding the potential risks of this treatment. STUDY FUNDING/COMPETING INTEREST(S): There was no funding sought or obtained for this study. K.T., V.T., B.V. and D.Z.-F. are employees or contractors to Monash IVF and hold a minority stock position in Monash IVF. R.J.W. reports no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Defining the limits of detection for chromosome rearrangements in the preimplantation embryo using next generation sequencing.

STUDY QUESTION: Is next generation sequencing (NGS) capable of detecting smaller sub-chromosomal rearrangements in human embryos than the manufacturer's quoted resolution suggests? SUMMARY ANSWER: NGS was able to detect unbalanced chromosome segments smaller than the manufacturer's resolution. WHAT IS KNOWN ALREADY: Array Comparative Genomic Hybridization (array-CGH) has been the gold standard platform used for PGD of chromosome rearrangements. NGS is a viable alternative to array-CGH for PGD of chromosome arrangements given that the manufacturer's guidelines quote a resolution of ≥20 Mb. However, as many patients carry a chromosome rearrangement <20 Mb, the detection limits of NGS warrant further investigation. STUDY DESIGN, SIZE, DURATION: This study involved a retrospective assessment of stored DNA samples from embryos that had previously been diagnosed as unbalanced by array-CGH as part of routine PGD in two separate IVF clinics between November 2013 and April 2017. SurePlex whole genome amplification (WGA) products derived from DNA extracted from an embryo biopsy sample known to carry an unbalanced form of a chromosome rearrangement were subjected to a specific NGS workflow (VeriSeq PGS). The results from the two technologies were compared for each sample. PARTICIPANTS/MATERIALS, SETTING, METHODS: WGA products from 200 embryos known to carry unbalanced rearrangements were sequenced and analysed. These embryos had been created by 75 patients known to carry a chromosome rearrangement (68 reciprocal translocations, 3 pericentric inversions, 1 paracentric inversion, 2 insertions and 1 dual reciprocal and inversion). Each sample was assessed for the size of the segmental gain/loss (Mb), copy number for each segment and chromosome, segregation pattern, the number of bins in the analysis software used and concordance with array-CGH results. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 294 unbalanced chromosome segments were assessed. NGS was capable of detecting 285/294 (97%) unbalanced segments previously identified using array-CGH. The final PGD diagnosis was concordant for 200/200 (100%) embryos. In total, 44/75 (59%) patients contained an unbalanced chromosome segment below the quoted 20 Mb manufacturer's stated resolution. Of these, 35/44 (80%) patients had segments that were able to be detected using NGS, whilst maintaining clinical outcome concordance. LIMITATIONS, REASONS FOR CAUTION: Our study subset did not include any rearrangements involving the Y chromosome. NGS has less available bins per chromosome compared to the array-CGH platform used, thus it remains possible that chromosome rearrangements predicted to be small but still detectable by array-CGH may not be feasible for testing using NGS. This should be considered when undertaking a theoretical feasibility assessment for detecting the chromosome rearrangement in question. Only one specific workflow for WGA and NGS was investigated in this study. WIDER IMPLICATIONS OF THE FINDINGS: This study has shown that NGS is available for the detection of unbalanced chromosome rearrangements ≥10 Mb. STUDY FUNDING/COMPETING INTEREST(S): Part sponsorship of the VeriSeq PGS kits used was provided by Illumina. The remainder of the kits were provided by two commercial IVF clinics. None of the authors has any conflicting interests to declare. TRIAL REGISTRATION NUMBER: N/A.

When two obese parents are worse than one! Impacts on embryo and fetal development.

The prevalence of overweight and obesity in reproductive-age adults is increasing worldwide. While the effects of either paternal or maternal obesity on gamete health and subsequent fertility and pregnancy have been reported independently, the combination of having both parents overweight/obese on fecundity and offspring health has received minimal attention. Using a 2 × 2 study design in rodents we established the relative contributions of paternal and maternal obesity on fetal and embryo development and whether combined paternal and maternal obesity had an additive effect. Here, we show that parental obesity reduces fetal and placental weights without altering pregnancy establishment and is not dependent on an in utero exposure to a high-fat diet. Interestingly combined parental obesity seemed to accumulate both the negative influences of paternal and maternal obesity had alone on embryo and fetal health rather than an amplification, manifested as reduced embryo developmental competency, reduced blastocyst cell numbers, impaired mitochondrial function, and alterations to active and repressive embryonic chromatin marks, resulting in aberrant placental gene expression and reduced fetal liver mtDNA copy numbers. Further understanding both the maternal cytoplasmic and paternal genetic interactions during this early developmental time frame will be vital for understanding how developmental programming is regulated and for the proposition of interventions to mitigate their effects.

Mitochondrial SIRT3 and its target glutamate dehydrogenase are altered in follicular cells of women with reduced ovarian reserve or advanced maternal age.

STUDY QUESTION: Is the activity of sirtuin 3 (SIRT3) altered in granulosa and cumulus cells from young women with reduced ovarian reserve or women of advanced maternal age? SUMMARY ANSWER: SIRT3 mRNA and active protein in granulosa and cumulus cells were decreased in women with reduced ovarian reserve and advanced maternal age. WHAT IS KNOWN ALREADY: Young women with reduced ovarian reserve or women of advanced maternal age have reduced oocyte viability, possibly due to altered granulosa and cumulus cell metabolism. The mitochondrial SIRT3 protein may be implicated in these processes as it is able to sense the metabolic state of the cell and alter mitochondrial protein function post-translationally. STUDY DESIGN, SIZE, DURATION: This is a prospective cohort study, in which women (n = 72) undergoing routine IVF/ICSI were recruited and allocated to one of three cohorts based on age and ovarian reserve (as assessed by serum anti-Mullerian hormone level). Women were classified as young (≤35 years) or of advanced maternal age (≥40 years). PARTICIPANTS/MATERIALS, SETTING, METHODS: Granulosa and cumulus cells were collected. SIRT3 mRNA and protein levels and protein activity was analysed in granulosa and cumulus cells via quantitative PCR, immunohistochemistry and western blotting, and deacetylation activity, respectively. Activity of the glutamate dehydrogenase (GDH) enzyme, a known target of SIRT3, was assessed, and acetylated proteins in mitochondria isolated from granulosa and cumulus cells were separated by immunoprecipitation and acetylation of GDH assessed by western blotting. Data for women with good prognosis (young women with normal ovarian reserve) were compared with those from young women with reduced ovarian reserve and those of advanced maternal age. MAIN RESULTS AND THE ROLE OF CHANCE: SIRT3 mRNA and active protein were present in granulosa and cumulus cells and co-localized to the mitochondria. SIRT3 mRNA in granulosa cells was decreased in young women with reduced ovarian reserve and advanced maternal age versus young women with normal ovarian reserve (P < 0.05). SIRT3 mRNA in cumulus cells was decreased in women of advanced maternal age versus young women with normal ovarian reserve only (P < 0.05). Granulosa cell GDH activity was decreased in young women with reduced ovarian reserve and in women of advanced maternal age (P < 0.05), whereas cumulus cell GDH activity was reduced in the advanced maternal age group only (P < 0.05). The acetylation profile of GDH in mitochondria revealed increased acetylation of GDH in granulosa and cumulus cells from women of advanced maternal age (P < 0.05) while young women with reduced ovarian reserve had increased GDH acetylation in granulosa cells only (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: Although patients were allocated to groups based on maternal age and ovarian reserve and matched for BMI, other maternal factors may also alter the 'molecular health' of ovarian cells. WIDER IMPLICATIONS OF THE FINDINGS: Our data suggest that SIRT3 post-translational modification of mitochondrial enzymes in human granulosa and cumulus cells may regulate GDH activity, thus altering the metabolic milieu surrounding the developing oocyte. Owing to the association between the decline in oocyte quality and pregnancy rates in women of advanced maternal age and the possible association with reduced ovarian reserve, knowledge of perturbed SIRT3 function in granulosa and cumulus cells may lead to novel therapies to improve mitochondrial metabolism in the oocyte and follicular cells in women undergoing IVF treatment. STUDY FUNDING/COMPETING INTEREST(S): No conflicts of interest to declare. Research was funded by an NHMRC project grant.