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Chemotherapy induced ovarian failure and infertility is an important concern in female cancer patients of reproductive age or younger, and non-invasive, pharmacological approaches to maintain ovarian function are urgently needed. Given the role of reduced nicotinamide adenine dinucleotide phosphate (NADPH) as an essential cofactor for drug detoxification, we sought to test whether boosting the NAD(P) + metabolome could protect ovarian function. We show that pharmacological or transgenic strategies to replenish the NAD + metabolome ameliorates chemotherapy induced female infertility in mice, as measured by oocyte yield, follicle health, and functional breeding trials. Importantly, treatment of a triple-negative breast cancer mouse model with the NAD + precursor nicotinamide mononucleotide (NMN) reduced tumour growth and did not impair the efficacy of chemotherapy drugs in vivo or in diverse cancer cell lines. Overall, these findings raise the possibility that NAD + precursors could be a non-invasive strategy for maintaining ovarian function in cancer patients, with potential benefits in cancer therapy., (© 2024. The Author(s).)

Females are born with a finite and non-renewable reservoir of oocytes, which therefore decline both in number and quality with advancing age. A striking characteristic of oocyte quality is that "ageing" effects manifest whilst women are in their thirties and are therefore still chronologically and physically young. Furthermore, this decline is unrelenting and not modifiable to any great extent by lifestyle or diet. Since oocyte quality is rate-limiting for pregnancy success, as the proportion of good-quality oocytes progressively deteriorate, the chance of successful pregnancy during each 6-12-month period also decreases, becoming exponential after 37 years. Unlike oocyte quality, age-related attrition in the size of the ovarian reservoir is less impactful for natural fertility since only one mature oocyte is typically ovulated per menstrual cycle. In contrast, oocyte numbers are pivotal for in-vitro fertilization success, since larger numbers enable better-quality oocytes to be found and is important for buffering the inefficiencies of the IVF process. The ageing trajectory is accelerated in ~10% of women, so-called premature ovarian ageing, with ~1% of women at the extreme end of this spectrum with loss of ovarian function occurring before 40 years of age, termed premature ovarian insufficiency. The aim of this review was to analyze how ageing impacts the size and quality of the oocyte pool along with emerging interventions for combating low oocyte numbers and improving quality.

Embryonic genome activation (EGA) marks the transition from dependence on maternal transcripts to an embryonic transcriptional program. The precise temporal regulation of gene expression, specifically the silencing of the Dux/murine endogenous retrovirus type L (MERVL) program during late 2-cell interphase, is crucial for developmental progression in mouse embryos. How this finely tuned regulation is achieved within this specific window is poorly understood. Here, using particle-tracking microrheology throughout the mouse oocyte-to-embryo transition, we identify a surge in cytoplasmic viscosity specific to late 2-cell interphase brought about by high microtubule and endomembrane density. Importantly, preventing the rise in 2-cell viscosity severely impairs nuclear reorganization, resulting in a persistently open chromatin configuration and failure to silence Dux/MERVL. This, in turn, derails embryo development beyond the 2- and 4-cell stages. Our findings reveal a mechanical role of the cytoplasm in regulating Dux/MERVL repression via nuclear remodeling during a temporally confined period in late 2-cell interphase., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Microtubules typically promote nuclear centring during early embryonic divisions in centrosome-containing vertebrates. In acentrosomal mouse zygotes, microtubules also centre male and female pronuclei prior to the first mitosis, this time in concert with actin. How nuclear centring is brought about in subsequent acentrosomal embryonic divisions has not been studied. Here, using time-lapse imaging in mouse embryos, we find that although nuclei are delivered to the cell centre upon completion of the first mitotic anaphase, the majority do not remain stationary and instead travel all the way to the cortex in a microtubule-dependent manner. High cytoplasmic viscosity in 2-cell embryos is associated with non-diffusive mechanisms involving actin for subsequent nuclear centring when microtubules again exert a negative influence. Thus, following the first mitotic division, pro-centring actin-dependent mechanisms work against microtubule-dependent de-centring forces. Disrupting the equilibrium of this tug-of-war compromises nuclear centring and symmetry of the subsequent division potentially risking embryonic development. This circuitous centring process exposes an embryonic vulnerability imposed by microtubule-dependent de-centring forces., (© 2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Inactivation of cyclin-dependent kinase 1 (Cdk1), controlled by cyclin B1 proteolysis, orders events during mitotic exit. Here, we used a FRET biosensor to study Cdk1 activity while simultaneously monitoring anaphase II and pronuclear (PN) formation in live mouse eggs throughout fertilization. We find that Cdk1 inactivation occurs over two phases separated by a 3-h pause, the first induces anaphase II and the second induces PN formation. Although both phases require the inhibitory Cdk1 kinase Wee1B, only the first involves cyclin B1 proteolysis. Enforcing the 3-h pause is critical for providing the delay required for male PN formation and is mediated by spindle midzone-dependent sequestration of Wee1B between the first and second phases. Thus, unlike continuous Cdk1 inactivation driven by cyclin B1 proteolysis during mitotic exit, MII oocytes engineer a physiologically important pause during fertilization involving two different pathways to inactivate Cdk1, only the first of which requires proteolysis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose of Review: Oocyte quality is rate-limiting for pregnancy success and declines with age. Here, I review animal-study evidence showing dramatic reversal of oocyte ageing with mitochondrial nutrients and explore clinical evidence related to their usage., Recent Findings: Oocyte ageing is strongly tied to mitochondrial dysfunction and oxidative stress. Quality-defining events occur over a protracted period (2-3 months in humans) when oocyte volume increases over 100-fold. Treating mice during the growth phase with mitochondrial modifiers such as CoQ10 combats oocyte ageing. Exciting new work shows that raising oocyte NAD+ levels also dramatically rejuvenate aged oocytes. However, evidence that any of these agents can reproducibly improve quality in humans is lacking. This is largely because there has been a focus on patients with poor ovarian response during IVF and/or low ovarian follicular pool size, rather than patients with poor oocyte quality. In addition, studies have used short-term treatment during ovarian stimulation after oocyte growth is already complete., Summary: Mitochondrial therapeutics such as NAD+-boosting used during the oocyte's growth phase markedly improve oocyte quality in mice. Evaluating them in humans should focus on patients with poor oocyte quality and utilise per-oocyte (rather than per-cycle) endpoints after adequate treatment that captures the growth phase when quality is defined., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Early decline in ovarian function known as premature ovarian aging (POA) occurs in around 10% of women and is characterized by a markedly reduced ovarian reserve. Premature ovarian insufficiency (POI) affects ~1% of women and refers to the severe end of the POA spectrum in which, accelerated ovarian aging leads to menopause before 40 years of age. Ovarian reserve refers to the total number of follicle-enclosed oocytes within both ovaries. Oocyte DNA integrity is a critical determinant of ovarian reserve since damage to DNA of oocytes within primordial-stage follicles triggers follicular apoptosis leading to accelerated follicle depletion. Despite the high prevalence of POA, very little is known regarding its genetic causation. Another little-investigated aspect of oocyte DNA damage involves low-grade damage that escapes apoptosis at the primordial follicle stage and persists throughout oocyte growth and later follicle development. Senataxin (SETX) is an RNA/DNA helicase involved in repair of oxidative stress-induced DNA damage and is well-known for its roles in preventing neurodegenerative disease. Recent findings uncover an important role for SETX in protecting oocyte DNA integrity against aging-induced increases in oxidative stress. Significantly, this newly identified SETX-mediated regulation of oocyte DNA integrity is critical for preventing POA and early-onset female infertility by preventing premature depletion of the ovarian follicular pool and reducing the burden of low-grade DNA damage both in primordial and fully-grown oocytes., Competing Interests: HH is a co-founder, shareholder, and advisor of Jumpstart Fertility Inc., which was founded to develop research into NAD+-dependent pathways involved in female fertility., (Copyright © 2021 Homer.)

Premature loss of ovarian activity before 40 years of age is known as primary ovarian insufficiency (POI) and occurs in ∼1% of women. A more subtle decline in ovarian activity, known as premature ovarian ageing (POA), occurs in ∼10% of women. Despite the high prevalence of POA, very little is known regarding its genetic causation. Senataxin (SETX) is an RNA/DNA helicase involved in repair of oxidative stress-induced DNA damage. Homozygous mutation of SETX leads to the neurodegenerative disorder, ataxia oculomotor apraxia type 2 (AOA2). There have been reports of POI in AOA2 females suggesting a link between SETX and ovarian ageing. Here, we studied female mice lacking either one (Setx+/-) or both (Setx-/-) copies of SETX over a 12- to 14-month period. We find that DNA damage is increased in oocytes from 8-month-old Setx+/- and Setx-/- females compared with Setx+/+ oocytes leading to a marked reduction in all classes of ovarian follicles at least 4 months earlier than typically occurs in female mice. Furthermore, during a 12-month long mating trial, Setx+/- and Setx-/- females produced significantly fewer pups than Setx+/+ females from 7 months of age onwards. These data show that SETX is critical for preventing POA in mice, likely by preserving DNA integrity in oocytes. Intriguingly, heterozygous Setx loss causes an equally severe impact on ovarian ageing as homozygous Setx loss. Because heterozygous SETX disruption is less likely to produce systemic effects, SETX compromise could underpin some cases of insidious POA., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Thyroid autoimmunity (TAI) - the presence of anti-thyroid peroxidase and/or anti-thyroglobulin antibodies - affects 8-14% of reproductively-aged women. It is hotly debated whether TAI adversely affects IVF/ICSI outcomes. This systematic review and meta-analysis evaluated the relationship between thyroid autoimmunity (TAI) and IVF/ICSI outcomes, both overall and amongst euthyroid women of known age using strict criteria for grouping pregnancy outcomes., Methods: The review was registered with PROSPERO: CRD42019120947. Searches were undertaken in MEDLINE, EMBASE, Web of Science and Cochrane Database from Inception-March 2020. Primary outcomes were clinical pregnancy rate, clinical miscarriage rate, biochemical pregnancy loss, livebirth rate per-cycle and live birth rate per clinical pregnancy (CP)., Results: 14 studies were included in the meta-analysis. Compared with women who tested negative for thyroid autoantibodies (TAI-), there was no significant difference in clinical pregnancy rate overall (OR 0.86; 95%CI [0.70, 1.05]; P = 0.14; 11 studies; I 2  = 29.0%), or in euthyroid women (OR 0.88; 95%CI [0.69, 1.12]; P = 0.29; 10 studies; I 2  = 32.0%). There was also no significant difference in clinical miscarriage rate overall (OR 1.04; 95%CI [0.52, 2.07]; P = 0.908; 8 studies; I 2  = 53%), or in euthyroid women (OR 1.18; 95%CI [0.52, 2.64]; P = 0.69; 7 studies; I 2  = 54%). There was no significant difference in biochemical pregnancy loss (OR 1.14; 95%CI [0.48, 2.72]; P = 0.769; 4 studies; I2 = 0.0%), live birth rate per cycle (OR 0.84; 95%CI [0.67, 1.06]; P = 0.145; I 2  = 1.7%), live birth rate per clinical pregnancy (OR 0.67; 95%CI [0.28, 1.60]; P = 0.369; I 2  = 69.2%), both overall and in euthyroid women as all studies included consisted of euthyroid women only. There was also no significant difference in number of embryos transferred, number of oocytes retrieved, mean maternal age or TSH levels overall or in euthyroid women., Conclusion: The findings of the present study suggest that thyroid autoimmunity has no effect on pregnancy outcomes in euthyroid women alone, or in euthyroid women and women with subclinical hypothyroidism.

The NAD + -dependent sirtuin deacetylase, Sirt1, regulates key transcription factors strongly implicated in ageing and lifespan. Due to potential confounding effects secondary to loss of Sirt1 function from the soma in existing whole-animal mutants, the in vivo role of Sirt1 in oocytes (oocyte-Sirt1) for female fertility remains unknown. We deleted Sirt1 specifically in growing oocytes and study how loss of oocyte-Sirt1 affects a comprehensive range of female reproductive parameters including ovarian follicular reservoir, oocyte maturation, oocyte mitochondrial abundance, oxidative stress, fertilization, embryo development and fertility during ageing. Surprisingly, eliminating this key sirtuin from growing oocytes has no effect in young females. During a 10-month-long breeding trial, however, we find that 50% of females lacking oocyte-Sirt1 become prematurely sterile between 9 and 11 months of age when 100% of wild-type females remain fertile. This is not due to an accelerated age-related decline in oocyte numbers in the absence of oocyte-Sirt1 but to reduced oocyte developmental competence or quality. Compromised oocyte quality does not impact in vivo oocyte maturation or fertilization but leads to increased oxidative stress in preimplantation embryos that inhibits cleavage divisions. Our data suggest that defects emerge in aged females lacking oocyte-Sirt1 due to concurrent age-related changes such as reduced NAD + and sirtuin expression levels, which compromise compensatory mechanisms that can cover for Sirt1 loss in younger oocytes. In contrast to evidence that increasing Sirt1 activity delays ageing, our data provide some of the only in vivo evidence that loss of Sirt1 induces premature ageing., (© 2020 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Meiotic divisions in oocytes are extremely asymmetric and require pre- and post-anaphase-onset phases of spindle migration. The latter induces membrane protrusion that is moulded around the spindle thereby reducing cytoplasmic loss. Here, we find that depleting the NAD biosynthetic enzyme, nicotinamide phosphoribosyl-transferase (Nampt), in mouse oocytes results in markedly longer spindles and compromises asymmetry. By analysing spindle speed in live oocytes, we identify a striking and transient acceleration after anaphase-onset that is severely blunted following Nampt-depletion. Slow-moving midzones of elongated spindles induce cortical furrowing deep within the oocyte before protrusions can form, altogether resulting in larger oocyte fragments being cleaved off. Additionally, we find that Nampt-depletion lowers NAD and ATP levels and that reducing NAD using small molecule Nampt inhibitors also compromises asymmetry. These data show that rapid midzone displacement is critical for extreme asymmetry by delaying furrowing to enable protrusions to form and link metabolic status to asymmetric division.

Mammalian oocytes rely heavily on mitochondrial oxidative phosphorylation (OXPHOS) for generating ATP. However, mitochondria are also the primary source of damaging reactive oxygen species (ROS). Mitochondrial de-regulation, therefore, underpins poor oocyte quality associated with conditions such as obesity and aging. The mitochondrial sirtuin, Sirt3, is critical for mitochondrial respiration and redox regulation. Interestingly, however, Sirt3 knockout (Sirt3 -/- ) mice do not exhibit systemic compromise under basal conditions, only doing so under stressed conditions such as high-fat diet (HFD)-induced obesity. Mouse oocytes depleted of Sirt3 exhibit increased ROS in vitro, but it is unknown whether Sirt3 is necessary for female fertility in vivo. Here, we test this for the first time by investigating ovarian follicular reserve, oocyte maturation (including detailed spindle assembly and chromosome segregation), and female fertility in Sirt3 -/- females. We find that under basal conditions, young Sirt3 -/- females exhibit no defects in any parameters. Surprisingly, all parameters also remain intact following HFD-induced obesity. Despite markedly increased ROS levels in HFD Sirt3 -/- oocytes, ATP levels nevertheless remain normal. Our data support that ATP is sustained in vivo through increased mitochondrial mass possibly secondary to compensatory upregulation of another sirtuin, Sirt1, which has overlapping functions with Sirt3., (© 2020 Federation of American Societies for Experimental Biology.)

In mitotic cells, DNA damage induces temporary G2 arrest via inhibitory Cdk1 phosphorylation. In contrast, fully grown G2-stage oocytes readily enter M phase immediately following chemical induction of DNA damage in vitro, indicating that the canonical immediate-response G2/M DNA damage response (DDR) may be deficient. Senataxin (Setx) is involved in RNA/DNA processing and maintaining genome integrity. Here we find that mouse oocytes deleted of Setx accumulate DNA damage when exposed to oxidative stress in vitro and during aging in vivo, after which, surprisingly, they undergo G2 arrest. Moreover, fully grown wild-type oocytes undergo G2 arrest after chemotherapy-induced in vitro damage if an overnight delay is imposed following damage induction. Unexpectedly, this slow-evolving DDR is not mediated by inhibitory Cdk1 phosphorylation but by APC-Cdh1-mediated proteolysis of the Cdk1 activator, cyclin B1, secondary to increased Cdc14B-dependent APC-Cdh1 activation and reduced Emi1-dependent inhibition. Thus, oocytes are unable to respond immediately to DNA damage, but instead mount a G2/M DDR that evolves slowly and involves a phosphorylation-independent proteolytic pathway., (© 2020 Subramanian et al.)

Reproductive aging in female mammals is an irreversible process associated with declining oocyte quality, which is the rate-limiting factor to fertility. Here, we show that this loss of oocyte quality with age accompanies declining levels of the prominent metabolic cofactor nicotinamide adenine dinucleotide (NAD + ). Treatment with the NAD + metabolic precursor nicotinamide mononucleotide (NMN) rejuvenates oocyte quality in aged animals, leading to restoration in fertility, and this can be recapitulated by transgenic overexpression of the NAD + -dependent deacylase SIRT2, though deletion of this enzyme does not impair oocyte quality. These benefits of NMN extend to the developing embryo, where supplementation reverses the adverse effect of maternal age on developmental milestones. These findings suggest that late-life restoration of NAD + levels represents an opportunity to rescue female reproductive function in mammals., Competing Interests: Declaration of Interests L.E.W., H.A.H., and D.A.S. are cofounders, shareholders, directors, and advisers of Jumpstart Fertility Inc., which was founded to develop the work described here, and are inventors on a patent (WO2019023748A1 “Methods to improve fertility”) that forms the basis of this work and has been licensed to Jumpstart Fertility. D.A.S. is an inventor on a patent (WO2013002880A1) that was previously licensed to Jumpstart Fertility. The salaries of M.J.B. and D.M.G. working in the labs of L.E.W. and R.B.G. at UNSW were paid by sponsored research from Jumpstart Fertility to UNSW. K.S. was an employee of Jumpstart Fertility. W.-H.J.H. and D.R.L. hold shares in Jumpstart Fertility. H.A.H. undertakes ART in the private sector in affiliation with Queensland Fertility Group. L.E.W. and D.A.S. are also advisers and shareholders in EdenRoc Sciences (Metro Biotech NSW, Metro Biotech, Liberty Biosecurity) and in Life Biosciences LLC and its daughter companies (Jumpstart Fertility, Continuum Biosciences, Senolytic Therapeutics, Selphagy, Animal Biosciences, Iduna). L.E.W. provides consulting work for Life Biosciences and is an adviser and shareholder in Intravital Pty Ltd. D.A.S. is also a founder, equity owner, adviser, director, consultant, investor, and/or inventor on patents licensed to Vium, Jupiter Orphan Therapeutics, Cohbar, Galilei Biosciences, Wellomics, EdenRoc Sciences (and affiliates Arc-Bio, Dovetail Genomics, Claret, Revere Biosciences, UpRNA, MetroBiotech, Liberty Biosecurity), Life Biosciences (and affiliates Selphagy, Senolytic Therapeutics, Spotlight Therapeutics, Animal Biosciences, Iduna, Continuum, Jumpstart Fertility, Iduna). D.A.S. is an inventor on a patent application filed by Mayo Clinic and Harvard Medical School that has been licensed to Elysium Health. For details see https://genetics.med.harvard.edu/sinclair., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Here we investigate whether the presence of germinal vesicle-stage oocytes (GV- oocytes) reflects poor oocyte developmental competence (or quality). This was a prospective, non-randomised, cohort pilot-study involving 60 patients undergoing in vitro fertilization/ intracytoplasmic sperm injection for whom complete pregnancy outcome data were available. Patients in whom GV- oocytes were retrieved (GV+) at transvaginal oocyte retrieval (TVOR) were compared with those from whom no GVs were retrieved (GV-). We found that GV+ ( n = 29) and GV- ( n = 31) patients were similarly aged (35.4 vs. 36.4 years; p = 0.446). GV+ patients had a mean of 2.41 ± 2.03 GVs and comparable yields of MII oocytes to GV- patients (11 ± 6.88 vs. 8.26 ± 4.84; p = 0.077). Compared with GV- patients, GV+ patients had markedly lower implantation rates (11.8% vs. 30.2%; p = 0.022) as well as oocyte utilisation rates for clinical pregnancy (2.3% vs. 6.8%; p = 0.018) and live-birth (1.9% vs. 5.7%; p = 0.029). DNA damage levels measured using γH2AX immunostaining were not different in oocytes from women <36 years versus those ≥36 years ( p = 0.606). Thus, patients who have GV- stage oocytes at TVOR exhibit poor oocyte quality reflected in reduced per-oocyte pregnancy success rates and uniformly high levels of oocyte DNA damage.

Infertility is described as unexplained when pregnancy does not occur despite ovulation, patent Fallopian tubes, and normal semen parameters. Oocyte developmental competence (or quality) is rate-limiting for pregnancy success as oocytes provide virtually all the cellular building blocks including mitochondria required during embryogenesis. However, available tests estimate oocyte numbers (anti-Müllerian hormone, follicle-stimulating hormone and antral follicle count) and ovulation (luteal phase serum progesterone) but not the third, and most pivotal, oocyte-specific parameter, quality. Severe depletion of the follicular reserve manifests as premature ovarian insufficiency and is an obvious cause of anovulation with overt symptoms and clear diagnostic criteria. In contrast, there are no biomarkers of poor oocyte quality other than through in vitro fertilization when readouts of oocyte quality such as preimplantation embryo development can be assessed. The most common cause of poor oocyte quality is natural aging, which is strongly tied to reduced oocyte mitochondrial efficiency and increased oxidative stress. In younger women, quality may also be impaired due to accelerated aging or sporadic genetic mutations which cause severe defects during oocyte and embryo development. Thus, poor oocyte quality often provides an explanation for infertility, but because it cannot be measured using conventional tests, many cases of infertility are often incorrectly labeled "unexplained." Since female age remains the best predictor of oocyte quality, age over 37 years should be considered an independent diagnostic criterion., Competing Interests: H.A.H. is a cofounder of a research company, JumpStart Fertility, whose intention is to develop agents for improving oocyte quality., (Thieme. All rights reserved.)

During mitosis, anaphase is triggered by anaphase-promoting complex (APC)-mediated destruction of securin and cyclin B1, which leads to inactivation of cyclin-dependent kinase 1 (Cdk1). By regulating APC activity, the mitotic spindle assembly checkpoint (SAC) therefore has robust control over anaphase timing to prevent chromosome mis-segregation. Mammalian oocytes are prone to aneuploidy, the reasons for which remain obscure. In mitosis, Cep55 is required post-anaphase for the final steps of cytokinesis. We found that Cep55-depleted mouse oocytes progress normally through early meiosis I, but that anaphase I fails as a result of persistent Cdk1 activity. Unexpectedly, Cdk1 inactivation was compromised following Cep55 depletion, despite on-time SAC silencing and intact APC-mediated proteolysis. We found that impaired Cdk1 inactivation was caused by inadequate inhibitory Cdk1 phosphorylation consequent upon failure to suppress Cdc25 phosphatase, identifying a proteolysis-independent step necessary for anaphase I. Thus, the SAC in oocytes does not exert exclusive control over anaphase I initiation, providing new insight into vulnerability to error., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Preimplantation genetic testing for aneuploidy (PGT-A) seeks to identify preimplantation embryos with a normal chromosome complement (euploid) during in vitro fertilisation (IVF). By sifting out embryos with abnormal chromosome numbers (aneuploid), PGT-A should theoretically improve pregnancy success. However, earlier versions of PGT-A were ineffective, and in some cases, detrimental, due to biopsy-induced trauma and because the technology at the time could analyse only a fraction of all chromosomes. More recently, the emergence of technologies enabling all chromosomes to be analysed and a switch to less traumatic blastocyst-stage biopsy have seen widespread uptake of PGT-A. Assessing the full impact of blastocyst biopsy PGT-A requires consideration of multiple factors, including embryonic mosaicism, sensitivity of the technological platform used, embryo loss during long-term in vitro culture, embryo cryopreservation and inter-clinic variability in expertise. Significantly, there hasnt yet been an appropriately designed randomised controlled trial (RCT) of blastocyst biopsy PGT-A analysed by intention-to-treat that accounts for all these parameters on a per-cycle basis. The three RCTs reporting benefits studied outcomes on a per-embryo transfer basis were small and underpowered and demonstrated benefits for a very select sub-group of good prognosis patients. The liberal use of this very expensive IVF add-on for other patient populations has not yet been shown to be effective, or indeed, without harm., (© 2019 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists.)

Recurrent miscarriage (RM), also known as recurrent pregnancy loss, is a distressing condition affecting around 1% of couples trying to conceive It can be very frustrating for both clinicians and patients as, despite intensive workup, no clear underlying pathology is forthcoming in at least 50% of couples. This leads to despair for patients and leaves clinicians at a loss for how to help. Desperation in both camps can promote the uptake of investigations and interventions of unproven benefit. The pathophysiology underpinning RM is incredibly diverse, involving areas such as haematology, endocrinology, immunology and genetics. During the seven to eight years since the UK Royal College of Obstetricians and Gynaecologists published guidelines on this topic in 2011, new evidence and guidance from expert authorities have emerged. Here, these important advances in this challenging field of clinical practice will be reviewed., (© 2018 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists.)

Understanding how human oocytes execute chromosome segregation is of paramount importance as errors in this process account for the overwhelming majority of human aneuploidies and increase exponentially with advancing female age. The spindle is the cellular apparatus responsible for separating chromosomes at anaphase. For accurate chromosome segregation, spindle microtubules must establish appropriately configured attachments to chromosomes via kinetochores. With regard to understanding the mechanistic basis for human aneuploidies therefore, it will be important to explore the molecular underpinnings of spindle structure and the interaction of its microtubules with chromosomes in human oocytes. Here we describe a technique for simultaneously immunolabelling chromosomes, spindle microtubules and kinetochores in human oocytes.

During mammalian mitosis, a proofreading network called the spindle assembly checkpoint (SAC) is indispensable for ensuring the fidelity of chromosome segregation. An inhibitory SAC signal is deputed to inhibits mitotic cell-cycle progression in response to misaligned chromosomes until such imperfections are rectified thereby ensuring equitable chromosome partitioning to daughter cells. Amongst the cast of SAC proteins, mitotic arrest deficient 2 (Mad2) plays a leading role in transducing the SAC signal. The aneuploidy and cancer predispositions of individuals who harbour genetic mutations in SAC genes emphasise the in vivo significance of this surveillance mechanism. In humans, congenital aneuploidies such as Down's syndrome demonstrate an exponential increase with advancing female age. Although largely the result of female meiosis I errors, the molecular entities that succumb with age in oocytes remain elusive. Declining oocyte SAC function could plausibly contribute to such errors. Until recently however, convincing evidence for a functional SAC in mammalian oocytes during meiosis I was unforthcoming. Here I review the evidence regarding the SAC in female mammalian meiosis I and how our understanding of this system has evolved in recent years. This review will focus on Mad2 as this is the SAC protein that has been most comprehensively investigated.

Mad2 is a pivotal component of the spindle assembly checkpoint (SAC) which inhibits anaphase promoting complex/cyclo-some (APC/C) activity by sequestering Cdc20 thereby regulating the destruction of securin and cyclin B. During mitosis, spindle depolymerisation induces a robust Mad2-dependent arrest due to inhibition of securin and cyclin B destruction. In contrast to mitosis, the molecular details underpinning the meiosis I arrest experienced by mouse oocytes exposed to spindle depolymerisation remain incompletely characterised. Notably, the role of Mad2 and the fate of the anaphase-marker, securin, are unexplored. As shown previously, we find that spindle depolymerisation by nocodazole inhibits first polar body extrusion (PBE) and stabilises cyclin B and cyclin-dependent kinase 1 activity in mouse oocytes. Here we show that stabilisation of cyclin B in nocodazole can be sustained for several hours and is associated with stabilisation of securin. These effects are SAC-mediated as, in oocytes depleted of the majority of Mad2 by morpholino antisense, securin and cyclin B are destabilised and 15% of oocytes undergo PBE. This reflects premature APC/C activation as a mutant form of cyclin B lacking its APC/C degradation signal is stable in Mad2-depleted oocytes. Moreover, homologues do not disjoin during the prolonged meiosis I arrest (> 18 h) induced by nocodaozole indicating that a non-cleavage mechanism is insufficient on its own for resolution of arm cohesion in mammalian oocytes. In conclusion, when all kinetochores lack attachment and tension, mouse oocytes mount a robust Mad2-dependent meiosis I arrest which inhibits the destruction of securin and cyclin B.

Although female meiosis I errors account for the majority of human aneuploidy, their molecular basis is largely unknown. By elucidating gene function, gene knockdown using RNA interference (RNAi) could shed light on this enigmatic process. In practice, however, the extreme paucity of immature human oocytes makes the evaluation of gene-targeting tools difficult. Here, we undertake RNAi in human oocytes and describe an approach employing mouse oocytes which could overcome the problem of limited biological material. We designed a short interfering RNA (siRNA) designated si539 to target the human mitotic arrest deficient 2 (hMad2) spindle checkpoint component. In human oocytes microinjected with si539, the hMad2 signal detected by Western blotting was 85-92% less intense than in oocytes injected with control siRNA indicating efficient silencing. Further examination of si539's targeting efficiency was undertaken using a green fluorescent protein (GFP)-tagged hMad2 mRNA construct in mouse oocytes. Consistent with Western blot analysis, si539 reduced hMad2-GFP expression in mouse oocytes by approximately 94% and relieved the meiosis I arrest otherwise induced by hMad2-GFP in mouse oocytes. By facilitating the investigation of candidate genes involved in regulating human female meiosis I, this approach can bring us closer to understanding the origins of aneuploidies such as Down's syndrome.

In mammalian somatic cells, the spindle assembly checkpoint (SAC) is indispensable for ensuring the fidelity of chromosome segregation by delaying cell-cycle progression in the face of even a single misaligned chromosome. In contrast, the role of the SAC in unperturbed mammalian oocytes is less well defined as progression through meiosis I is unaltered in mouse oocytes in the presence of one or a few misaligned chromosomes. Furthermore, attempts to disable the function of the SAC protein, Mad2, in mouse oocytes have produced conflicting results. To gain further insight into SAC function during female mammalian meiosis I, we recently utilised a morpholino-based antisense approach to deplete the majority of Mad2 in mouse oocytes. Our results define a clear role for Mad2 in ensuring the proper timing of meiosis I events and ultimately, in ensuring the fidelity of homologue disjunction. We discuss the implications of these results for the regulation of meiosis I in mammalian oocytes and for the genesis of human aneuploidy.

In mitosis, the spindle checkpoint protein Mad2 averts aneuploidy by delaying anaphase onset until chromosomes align. Here we show that depletion of Mad2 in meiosis I mouse oocytes induced an increased incidence of aneuploidy. Proteolysis of cyclin B and securin commenced earlier in Mad2-depleted oocytes, resulting in a shortened duration of meiosis I. Furthermore, overexpression of Mad2 inhibited homolog disjunction. We conclude that Mad2 delays the onset of cyclin B and securin degradation and averts aneuploidy during meiosis I in mammalian oocytes. The data suggest a link between trisomies such as Down syndrome and defective oocyte spindle checkpoint function.

Objective: To review the literature on the diagnosis, prevalence, and treatment of the septate uterus, with special reference to hysteroscopic metroplasty and its effect on reproductive outcome., Design: Pertinent studies were identified through a computer MEDLINE search. References of selected articles were hand-searched for additional citations., Result(s): Reliable diagnosis of the septate uterus depends on accurate assessment of the uterine fundal contour. At present, the combined use of laparoscopy and hysteroscopy is the gold standard for diagnosis, although recent reports of two-dimensional, transvaginal, contrast ultrasound and of three-dimensional ultrasound appear promising. The prevalence of the septate uterus is increased in women with repeated pregnancy loss. A metaanalysis of published retrospective data comparing pregnancy outcome before and after hysteroscopic septoplasty indicated a marked improvement after surgery., Conclusion(s): The hysteroscopic approach to treatment, with its simplicity, minimal postoperative sequelae, and improved reproductive outcome, has enabled a more liberalized approach to treatment that is now being extended to include not only patients with recurrent pregnancy loss and premature labor but also patients with infertility, especially if IVF is being contemplated.