Two-step nuclear centring by competing microtubule- and actin-based mechanisms in 2-cell mouse embryos.

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.

First meiotic anaphase requires Cep55-dependent inhibitory cyclin-dependent kinase 1 phosphorylation.

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.

Sirt3 is dispensable for oocyte quality and female fertility in lean and obese mice.

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.

Understanding oocyte ageing: can we influence the process as clinicians?

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.

Thyroid autoimmunity and IVF/ICSI outcomes in euthyroid women: a systematic review and meta-analysis.

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; I2 = 29.0%), or in euthyroid women (OR 0.88; 95%CI [0.69, 1.12]; P = 0.29; 10 studies; I2 = 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; I2 = 53%), or in euthyroid women (OR 1.18; 95%CI [0.52, 2.64]; P = 0.69; 7 studies; I2 = 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; I2 = 1.7%), live birth rate per clinical pregnancy (OR 0.67; 95%CI [0.28, 1.60]; P = 0.369; I2 = 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.

Disruption of Spermatogenesis and Infertility in Ataxia with Oculomotor Apraxia Type 2 (AOA2).

Ataxia with oculomotor apraxia type 2 (AOA2) is a rare autosomal recessive cerebellar ataxia characterized by onset between 10 and 20 years of age and a range of neurological features that include progressive cerebellar atrophy, axonal sensorimotor neuropathy, oculomotor apraxia in a majority of patients, and elevated serum alpha-fetoprotein (AFP). AOA2 is caused by mutation of the SETX gene which encodes senataxin, a DNA/RNA helicase involved in transcription regulation, RNA processing, and DNA maintenance. Disruption of senataxin in rodents led to defective spermatogenesis and sterility in males uncovering a key role for senataxin in male germ cell survival. Here, we report the first clinical and cellular evidence of impaired spermatogenesis in AOA2 patients. We assessed sperm production in three AOA2 patients and testicular pathology in one patient and compared the findings to those of Setx-knockout mice. Sperm production was impaired in all patients assessed (3/3, 100%). Analyses of testicular biopsies from an AOA2 patient recapitulate features of the histology seen in Setx-knockout mice, strongly suggesting an underlying mechanism centering on DNA-damage-mediated germ cell apoptosis. These findings support a role for senataxin in human reproductive function and highlight a novel clinical feature of AOA2 that extends the extra-neurological roles of senataxin. This raises an important reproductive counseling issue for clinicians, and fertility specialists should be aware of SETX mutations as a possible diagnosis in young male patients presenting with oligospermia or azoospermia since infertility may presage the later onset of neurological manifestations in some individuals.

Preimplantation genetic testing for aneuploidy (PGT-A): The biology, the technology and the clinical outcomes.

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.

Modern management of recurrent miscarriage.

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.

Spindle assembly checkpoint signalling is uncoupled from chromosomal position in mouse oocytes.

The spindle assembly checkpoint (SAC) averts aneuploidy by coordinating proper bipolar chromosomal attachment with anaphase-promoting complex/cyclosome (APC/C)-mediated securin and cyclin B1 destruction required for anaphase onset. The generation of a Mad2-based signal at kinetochores is central to current models of SAC-based APC/C inhibition. During mitosis, kinetochores of polar-displaced chromosomes, which are at greatest risk of mis-segregating, recruit the highest levels of Mad2, thereby ensuring that SAC activation is proportionate to aneuploidy risk. Paradoxically, although an SAC operates in mammalian oocytes, meiosis I (MI) is notoriously error prone and polar-displaced chromosomes do not prevent anaphase onset. Here we find that Mad2 is not preferentially recruited to the kinetochores of polar chromosomes of wild-type mouse oocytes, in which polar chromosomes are rare, or of oocytes depleted of the kinesin-7 motor CENP-E, in which polar chromosomes are more abundant. Furthermore, in CENP-E-depleted oocytes, although polar chromosomal displacement intensified during MI and the capacity to form stable end-on attachments was severely compromised, all kinetochores nevertheless became devoid of Mad2. Thus, it is possible that the ability of the SAC to robustly discriminate chromosomal position might be compromised by the propensity of oocyte kinetochores to become saturated with unproductive attachments, thereby predisposing to aneuploidy. Our data also reveal novel functions for CENP-E in oocytes: first, CENP-E stabilises BubR1, thereby impacting MI progression; and second, CENP-E mediates bi-orientation by promoting kinetochore reorientation and preventing chromosomal drift towards the poles.

Understanding oocyte ageing.

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.

A surge in cytoplasmic viscosity triggers nuclear remodeling required for Dux silencing and pre-implantation embryo development.

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.

Fertility treatment pathways and births for women with and without polycystic ovary syndrome-a retrospective population linked data study.

OBJECTIVE: To study the fertility treatment pathways used by women with and without polycystic ovary syndrome (PCOS) and which pathways were more likely to result in a birth. DESIGN: This retrospective national community-based cohort study used longitudinal self-report survey data (collected 1996-2022; aged 18-49 years) from women born in 1973-1978 who are participants in the Australian Longitudinal Study on Women's Health. The study also used linked administrative data on fertility treatments (1996-2021). PATIENTS: Of the 8,463 eligible women, 1,109 accessed fertility treatment and were included. EXPOSURE: Polycystic ovary syndrome diagnosis was self-reported. MAIN OUTCOME MEASURE: use of ovulation induction (OI), intrauterine insemination, and/or in vitro fertilization (IVF) was established through linked administrative data. Births were self-reported. RESULTS: One in 10 of the eligible participants had PCOS (783/7,987, 10%) and 1 in 4 of the women who used fertility treatment had PCOS (274/1,109, 25%). Women with PCOS were 3 years younger on average at first fertility treatment (M = 31.4 years, SD = 4.18) than women without PCOS (M = 34.2 years, SD = 4.56). Seven treatment pathways were identified and use differed by PCOS status. Women with PCOS were more likely to start with OI (71%; odds ratio [OR] 4.20, 95% confidence interval [CI]: 2.91, 6.07) than women without PCOS (36%). Of the women with PCOS who started with OI, 46% required additional types of treatment. More women without PCOS ended up in IVF (72% vs. 51%). Overall, 63% (701/1,109) had an attributed birth, and in adjusted regressions births did not vary by last type of treatment (IVF: 67%, reference; intrauterine insemination: 67%, OR 0.94 95% CI: 0.56, 1.58; OI: 61%, OR 0.71, 95% CI: 0.52, 0.98), or by PCOS status (OR 1.27, 95% CI: 0.91, 1.77). By age, 74% of women under 35 years (471/639) and 49% of women 35 years or older had a birth. CONCLUSION: More women with PCOS used fertility treatment but births were equivalent to women without PCOS. Most women followed clinical recommendations. Births did not differ between pathways, so there was no disadvantage in starting with less invasive treatments (although there may be financial or emotional disadvantages).

Fertility protection during chemotherapy treatment by boosting the NAD(P)+ metabolome.

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.

The APC/C in female mammalian meiosis I.

The anaphase-promoting complex or cyclosome (APC/C) orchestrates a meticulously controlled sequence of proteolytic events critical for proper cell cycle progression, the details of which have been most extensively elucidated during mitosis. It has become apparent, however, that the APC/C, particularly when acting in concert with its Cdh1 co-activator (APC/C(Cdh1)), executes a staggeringly diverse repertoire of functions that extend its remit well outside the bounds of mitosis. Findings over the past decade have not only earmarked mammalian oocyte maturation as one such case in point but have also begun to reveal a complex pattern of APC/C regulation that underpins many of the oocyte's unique developmental attributes. This review will encompass the latest findings pertinent to the APC/C, especially APC/C(Cdh1), in mammalian oocytes and how its activity and substrates shape the stop-start tempo of female mammalian first meiotic division and the challenging requirement for assembling spindles in the absence of centrosomes.

The oocyte spindle midzone pauses Cdk1 inactivation during fertilization to enable male pronuclear formation and embryo development.

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.

Homologue disjunction in mouse oocytes requires proteolysis of securin and cyclin B1.

Disjunction of pairs of homologous chromosomes during the first meiotic division (MI) requires anaphase-promoting complex (APC)-mediated activation of separase in budding yeast and Caenorhabditis elegans, but not Xenopus laevis. It is not clear which model best fits the mammalian system. Here we show that homologue disjunction in mouse oocytes is dependent on proteolysis of the separase inhibitor securin and the Cdk1 regulatory sub-unit cyclin B1. Proteolysis of both proteins was entirely dependent on their conserved destruction box (D-box) motifs, through which they are targeted to the APC. These data indicate that the mechanisms regulating homologue disjunction in mammalian oocytes are similar to those of budding yeast and C.elegans.

Senataxin: A New Guardian of the Female Germline Important for Delaying Ovarian Aging.

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.