Reproductive mechanisms, pathologies, and health inclusivity: insights from the 2023 Annual Meeting of the Society for Reproductive Biology

Speakers: Jose Polo, Patrick Western, Gary Hime, and Margot Day

Understanding the intricate cellular mechanisms underlying reproductive function is critical for advancing both basic and applied sciences in reproductive biology. This section explores various model systems and innovative methodologies that have significantly contributed to the field. Notably, several research groups are breaking new ground in probing the cellular foundations of reproductive biology, covering both established and novel model systems, untangling developmentally important genes as vehicles for epigenetic influences, and the translational implications of modifying embryo culture conditions.

Model systems are instrumental in understanding the physiology of reproduction, and the vinegar fly, Drosophila melanogaster, has been a laboratory model organism for over 100 years. Genome sequencing has identified numerous human genetic variants that have been linked to infertility (Nagirnaja et al. 2022; Oud et al. 2022), and many of the affected human genes are conserved in Drosophila. With the ability to rapidly mutate any of the 15,000 genes in the fly genome, researchers can exploit genetic tools to conduct sophisticated experiments relating to gene function. Professor Gary Hime (University of Melbourne) has pioneered the use of such tools in the field of male reproduction, and has illustrated the versatility of Drosophila models, which have improved understanding of several RNA-binding proteins, transcription factors, and signalling molecules in regulating male fertility (Brill et al. 2000). In addition, Drosophila testes offer an ideal model system for studying stem cell regulation due to the discrete identity and position of the germline stem cells and neighbouring daughter cells, in contrast to mammalian testes where these populations are less distinct (de Cuevas and Matunis 2011). To this end, Professor Hime’s team showed that loss of function of the RNA-binding protein, Musashi (Msi), resulted in loss of germline stem cells and premature germ cell differentiation, indicating that Msi is required for the maintenance of stem cell identity (Siddall et al. 2006). More recently, Drosophila models have been used as a tool to dissect the function of human genetic variants linked with reproductive phenotypes. For example, the gene SART3 encodes an RNA-binding protein that is critical to spliceosome function (Whitmill et al. 2016). Bi-allelic variants of SART3 were shown to underlie a spliceosomopathy, a syndrome characterised by gonadal dysgenesis and a range of neurodevelopmental comorbidities (Ayers et al. 2023). Attempts to generate models of SART3 variants in both zebrafish and mice result in embryonic lethality (Trede et al. 2007; Whitmill et al. 2016). However, knockdown of the Drosophila orthologue of SART3, Rnp4f, resulted in disrupted testicular development and neuronal defects. This identified a conserved role of Rnp4f in testis development, where it is required in somatic gonadal cells for spermatogenesis (Ayers et al. 2023). This demonstrates the utility of the Drosophila model for the study of specific aspects of reproductive biology, such as mutations with known embryonic lethality in other species.

A very different novel model system was introduced by Professor Jose Polo (University of Adelaide): the iBlastoid, a three-dimensional model of a blastocyst. Since John Gurdon’s seminal experiments in 1958 (Gurdon et al. 1958), biologists have known that every cell in the body carries the information and potential to make any cell type. A major development in the understanding of pluripotency came with discovery of the Yamanaka factors, OCT4, SOX2, KLF4, and cMYC, which facilitated the advent of induced pluripotent stem cells (iPSCs) – cells able to differentiate into any cell type of the body, much like embryonic stem cells derived from the epiblast of blastocysts (Takahashi et al. 2007). Recently, Professor Polo and his team have made significant progress towards understanding the molecular mechanisms that underpin the transitions into primed or naïve states in human iPSCs. Molecular reprogramming trajectories were reconstructed using single cell transcriptomics, revealing that reprogramming into the primed and naïve human pluripotency states follow diverging and distinct trajectories (Liu et al. 2020). Trophectoderm (TE) lineage-associated transcription factors had unexpected roles throughout reprogramming and a transient TE-like signature was observed in a subpopulation of TE cells during this time (Liu et al. 2020). Notably, stabilising the TE-like state enabled induced trophoblast stem cells (iTSCs) to be derived. Professor Polo described several recent applications of iTSCs and human induced pluripotent stem (hiPS) cells. iTSCs were used to develop an in vitro model of placental cell types that was used to understand the mechanisms of vertical transfer of SARS-CoV2 infection (Chen et al. 2023). Meanwhile, reprogramming intermediates maintained a primitive endoderm-like signature in vitro, beyond the epiblast and TE signatures, and remarkably, formed three-dimensional blastocyst-like structures termed iBlastoids (Liu et al. 2020). Following extensive characterisation of iBlastoids, the team confirmed similar morphological, molecular, and functional similarities to blastocysts and thus presented an exciting new model to study the early stages of embryonic development (Liu et al. 2020; Tan et al. 2024).

Epigenetic modifications underpin many of the connections between environmental insults and effects on offspring development, but how these signatures are encoded, and the mechanisms involved, are poorly understood. Professor Patrick Western (Monash University) and his team investigated the role of Polycomb repressive complex 2 (PRC2), which catalyses the epigenetic modification H3K27me3 to repress developmental genes in many tissues. In mice, oocyte-specific deletion of the Eed gene, encoding a core PRC2 subunit, de-repressed (allowed expression of) >300 genes involved in an array of developmental processes (Jarred et al. 2022). Offspring from these oocytes were developmentally delayed and exhibited placental hyperplasia. They also underwent accelerated growth late in fetal life, extended gestation, and perinatal overgrowth, compared to genetically identical controls (Prokopuk et al. 2018; Oberin et al. 2024). Notably, fetal development was significantly affected as demonstrated by altered bone and brain development, and behaviour, in offspring. Whether these findings translate to human development remains to be seen. However, many of these genes contained H3K27me3 in human germinal vesicle oocytes, suggesting that PRC2 activity is conserved in human oocytes. Understanding the processes of epigenetic regulation is critical for determining how epigenetic programming in oocytes regulates health and development of the next generation and by extension how environmental stress alters non-genetic inheritance in mammals.

Embryos generated in vitro show evidence of altered cellular processes and cell composition, which impact embryo viability. Given the discrepancies between the amino acid (AA) composition of common embryo culture media and the composition of fluids from the reproductive tract, the mechanism of action of individual AAs is difficult to dissect in vitro. Associate Professor Margot Day’s team (University of Sydney) developed AA-free embryo culture media (‘no AA media’) to systematically evaluate the impacts of individual AAs on embryo development beyond roles in protein production. Key findings indicate that supplementation with either L-glutamine or L-proline improved blastocyst development rates and hatching but had no effect on blastocyst cell numbers compared to the ‘no AA media’. Further work indicated that, when L-proline was supplemented during IVF or from the late two-cell stage during embryo culture, embryo development rates improved (Morris et al. 2020; Treleaven et al. 2021). Proline is a conditionally essential AA with diverse cellular functions. To dissect the possible mechanism of action by which L-proline improved embryo development rate, Associate Professor Day’s team investigated proline transport in oocytes and embryos. Of the proline transporters expressed in oocytes and embryos, only B⁰AT1 (SLC6A19) was expressed at the plasma membrane of zygotes and blastomeres of all preimplantation stage embryos, specifically, at surfaces that were not in contact with other blastomeres (Treleaven et al. 2022). In Slc6a19^−/− mice, decreased litter sizes and embryo development rates compared to wild-type animals indicated that optimal proline uptake is essential for normal fertility (Treleaven et al. 2022). However, proline transporters including B⁰AT1 also transport other AAs. The addition of several other AAs (including phenylalanine, glycine, alanine, betaine, histidine, and sarcosine) reduced proline uptake in oocytes. Moreover, addition of these AAs to ‘no AA media’ eliminated the beneficial effect of L-proline supplementation on blastocyst formation rate (Morris et al. 2020). Proline contributes to several cellular functions, some of which were investigated further to understand the mechanism of action. Media osmolality was negatively associated with zygote development to the blastocyst stage; however, supplementation with L-proline (and other AAs) rescued blastocyst rates at high osmolality (330 mOsm) (Morris et al. 2020). Likewise, L-proline positively modulated growth factor-like signalling and protein biosynthesis pathways that improved blastocyst formation rate (Morris et al. 2020). Importantly, inhibition of proline metabolism reverted the positive effects of L-proline on blastocyst formation and additional experiments revealed that proline also reduced production of reactive oxygen species (Hardy et al. 2023). These studies have demonstrated that proline is a small molecule with complex functions and highlighted the importance of improving understanding of the broader roles of AAs in oocyte and embryo development to enhance culture media formulation and embryo development outcomes in vitro.

Collectively, this session illustrated key advancements in diverse approaches to unravel the mechanisms of reproductive processes. The research highlighted herein underscores the significance of model organisms and novel biological tools in providing insights into genetic, cellular, and molecular dynamics that are essential for fertility and development. Importantly, this research provided key examples of fundamental biology questions leading to translational applications, from improving assisted reproductive technologies to understanding developmental disorders, paving the way for future research and therapeutic strategies in reproductive health.

Speakers: Jacinta Martin, Angela Lees, and Deidre Mattiske

Human activity has irreversibly changed many aspects of the environment worldwide, notably including a changing global climate and systemic pollution by numerous chemical agents. These environmental changes profoundly affect the health and fertility of our species, including future generations, impact animals used for food and agriculture, and endanger biodiversity. This symposium session highlighted how the economy, health, and fertility can be adversely affected by chemical exposure and heat stress that have arisen due to uncontrolled human activity.

In virtually all developed and numerous undeveloped countries, humans are continuously being exposed to environmental contaminants, with one of the most widespread groups being per-fluoroalkyl and polyfluoroalkyl substances (PFAS). PFAS are a diverse group of highly fluorinated synthetic chemicals that are very stable and extremely persistent in the environment (Buck et al. 2011). Due to their heat- and water-resistant properties, PFAS have been used in a variety of products such as firefighting foams, non-stick pans and other cookware, artificial turf and water-repellent clothing, since the 1950s (Buck et al. 2011; Gaines 2023). In recent decades, PFAS exposure has been associated with a variety of negative health effects including altered immune function, dysregulated lipid and insulin activity, adverse reproductive outcomes and cancer (Calvert et al. 2021; Fenton et al. 2021). Such effects are exacerbated by the bioaccumulating and water-soluble properties of PFAS, as well as by transmission to the next generation through the placenta, blood, and breastmilk (Kurwadkar et al. 2022; Criswell et al. 2023). However, the impact of PFAS exposure across critical developmental periods in offspring remains unknown. To understand the impact of gestational and postnatal PFAS exposure on offspring development, Dr Jacinta Martin (University of Newcastle) administered a cocktail of nine PFAS chemicals to female mice through drinking water. The mice were exposed from the periconception period, throughout pregnancy, and offspring were exposed from birth and throughout weaning until adulthood. This cocktail mimicked the composition and concentration of PFAS found in groundwater in a PFAS-contaminated site in Williamtown, NSW, Australia (Calvert et al. 2024). Despite having comparable litter sizes at late gestation, female mice exposed to PFAS had smaller litter sizes shortly after birth compared to control mice, suggestive of an infanticide effect. Infanticide is a common behaviour of parental mice when unhealthy young are identified. Alarmingly, maternal PFAS exposure also led to endocrine and metabolic dysfunction in offspring before weaning, and behavioural changes at adulthood (Deidre Mattiske, unpubl. data). Collectively, these findings demonstrate that exposure to environmental PFAS during early life, including throughout pregnancy and via lactation, has a substantial influence on multiple aspects of offspring life-long health. Further investigation is now warranted to determine the extent of these PFAS-mediated impacts in humans, which will build a strong case for better regulation of PFAS use and thus the reduction of daily exposure (Calvert et al. 2021).

In addition to PFAS, humans are exposed to numerous other endocrine-disrupting chemicals (EDCs), which contributed to a staggering economic burden of USD340 billion in the USA and USD217 billion in Europe in 2010 (Attina et al. 2016). EDCs are natural or synthetic chemicals that interfere with the endocrine system and cause an adverse health effect (Diamanti-Kandarakis et al. 2009; Schjenken et al. 2021). Like PFAS, some EDCs were used for a variety of purposes but are now recognised for their detrimental impact on health. For instance, diethylstilbesterol (DES) is a synthetic form of oestrogen that was widely used as a pharmaceutical treatment in the 1940–1970s to prevent pregnancy complications including miscarriage and preterm birth. Over 10 million people are estimated to have been exposed to DES (Reed and Fenton 2013). Contrary to its intended purpose, DES does not prevent miscarriage and, instead, exposure leads to an increased incidence of cancer and reproductive abnormalities in daughters. DES exposure also increases risk for hypospadias in sons, a birth defect where the urethra fails to develop at the tip of the penis (Mattiske and Pask 2021). As the effects of DES are predicted to be transgenerational, the chance for early corrective treatment for many people has been lost. Further, the consequences on human health past the second generation of exposure are currently unknown. To investigate the effects of gestational DES exposure over multiple generations, Dr Deidre Mattiske (University of Melbourne) treated pregnant female mice (F0 generation) with DES during late gestation and examined reproductive system development and fertility in male and female offspring across four generations (F1–F4 generations). Male DES offspring were more susceptible to hypospadias and exhibited an increase in severity, and a reduction in anogenital distance was also evident, at least in the F1–F3. In some DES F3-F4 male offspring, fertility was impaired because they were unable to form copulatory plugs and their sperm progressive motility and velocity (F1–F4), key measures of sperm function, were reduced. Similarly, 100% of female DES F1 offspring had a urethra-vaginal fistula. Vaginal opening occurred precociously in DES female offspring through to the F4 generation, indicating early onset of puberty. Furthermore, in F1 and F2 offspring, folliculogenesis was altered, with more secondary follicles and less primordial follicles. Alarmingly, the F0 mice were only treated with a single dose and type of EDC, and yet, transgenerational effects were observed for both males and females that persisted through to the F4 generation. This study has implications for the millions of people affected by DES, including future generations. These findings highlight the pressing need to improve understanding of the transgenerational effects of EDC exposure and develop appropriate interventions.

Human activities have exacerbated climate change, and the impact of subsequent heat stress on livestock species is of urgent economic and welfare concern (Polsky and von Keyserlingk 2017; van Wettere et al. 2021). Thermal exchange mechanisms in livestock are influenced by numerous factors including climate, shade infrastructure, and physiology (DeShazer et al. 2009). Without cool periods to allow for the regulation of body temperature, there will likely be an increased scrotal temperature in bulls that is profoundly detrimental to sperm quality. Specifically, heat stress in bulls causes decreased sperm concentration and motility, and increased morphological abnormalities and DNA damage (Kastelic et al. 2018). Our current understanding of how heat stress affects bull fertility is largely derived from studies of scrotal insulation rather than whole-body heat stress. The scrotal insulation approach, however, renders bulls unable to fully utilise their normal thermoregulatory mechanisms to mitigate the reproductive impact of heat stress (Kastelic et al. 2018) and may not be truly representative. Scrotal insulation directly influences the ability of the scrotum to leverage scrotal sweat glands and the counter-current heat exchange that exists through the testicular artery and venous pampiniform plexus. To improve the model, Dr Angela Lees (University of Queensland) and team surgically inserted data loggers into the flank and scrotum of bulls to enable simultaneous and continual temperature monitoring of the body and scrotum. While other studies have surgically inserted data loggers at a lower point in the scrotum, Dr Lees’ team instead attached data loggers to the parietal tunica vaginalis at the midway point of the testis (Wallage et al. 2017). This technique previously demonstrated consistent regulation of scrotal temperature despite greater variation in body temperature during housing in a paddock versus individual pens (Wallage et al. 2017). More recently, the team reported that when bulls were exposed to acute (5 days) or chronic (14 days) simulated heat waves, their thermoregulatory mechanisms could not maintain a lower intrascrotal temperature (Wallage et al. 2022). Ongoing work includes surgical refinement, assessing sperm morphology, characterising the recovery period, and simulating repeated heat waves. The effects of heat stress on male fertility take 1–2 weeks to become evident in the bull ejaculate (Meyerhoeffer et al. 1985), and reproductive function recovery can take up to 10–11 weeks (Garcia-Oliveros et al. 2022). Thus, intervals between natural heat waves may be insufficient for bulls to complete a full spermatogenesis cycle of 8.7 weeks and the additional ~2 weeks required for epididymal sperm maturation. Concerningly, the effects of repeated exposures on the magnitude of negative semen quality remain to be tested. Intrascrotal data loggers, however, have now been tested in rams (Shahat et al. 2020), and in bulls they have demonstrated beneficial contributions of voluntary use of shade structures on intrascrotal cooling and subsequent sperm quality (Shahat et al. 2023). This methodology can inform livestock management practices during heat waves, and identify bulls with enhanced scrotal thermoregulatory capacity, thereby improving the efficiency of livestock industries.

Collectively, these examples demonstrate how human actions can impact the environment in ways that affect reproductive capacity and the health and development of future generations in both humans and livestock. Unfortunately, the associated health and economic burdens have not been clearly assessed and will worsen without adequate intervention. Overall, this session highlighted a clear need for a better understanding of environmental impacts in order to develop treatments or interventions to mitigate these adverse effects.

Speakers: Ada Cheung, Ken Pang, and Patrick Thomsen

Although inclusivity in reproductive health is a fundamental right, it is not the reality for many. This symposium explored approaches to ensure inclusivity in reproductive health research and clinical care, with insights from research focused on the reproductive health of transgender (trans) people, and Pacific Rainbow+ communities in Aotearoa, New Zealand. During each talk and the ensuing panel discussion, speakers provided valuable insights into underlying gaps in knowledge by generously sharing their teams’ experiences in undertaking research with these communities. Existing practices in the field of reproductive research were challenged, with reflections on what is required to ensure inclusivity in reproductive health. This symposium was especially timely with numerous anti-trans movements across North America, with the aim to restrict gender-affirming medical care. At the same time, abortion-restriction bills are even more prevalent, preventing women from accessing safe and reliable terminations, even in situations where the pregnancy is of significant threat to the life of the mother.

At least 1–5% of the population identifies as trans or non-binary but a significant portion of these people experience barriers to health treatments and face discrimination. Most importantly, trans health has only recently emerged as an area of study, with very few clinical trials and systematic reviews performed on trans health. Associate Professor Ada Cheung, a researcher at the University of Melbourne and Austin Health, is advocating for better research into trans health with the ultimate goal of improving access to medical services for trans people. Three major areas for improvement have been identified: mental health and wellbeing; health service delivery; and gender-affirming hormone therapy (GAHT). Sustained progress in these areas requires the co-design of clinical trials and approaches with members of the trans and non-binary communities, improved science communication, and better training for healthcare providers and doctors. Notably, work by Associate Professor Cheung and team has revealed providing trans men with gender-affirming hormone treatment (testosterone) significantly improves mental health and lowers suicidal ideation. In trans men and non-binary people using testosterone therapy, oophorectomy reduces levels of estradiol, a major hormonal regulator of bone density. Studies in mice have demonstrated that co-treatment with testosterone and estradiol prevented a loss in bone density in ovariectomised mice compared with testosterone alone, highlighting the importance of estradiol (and ovarian aromatase activity) in maintaining bone density (Goetz et al. 2017). Future research should continue to assess the consequences of oophorectomy on long-term health (Kumar et al. 2022). Leaving ovaries in situ, where possible, will likely minimise any health impacts. Finally, the potential for pregnancy in trans men was discussed, but the optimal timing for cessation of testosterone prior to pregnancy is less clear. Case reports have described successful oocyte retrieval after ceasing testosterone treatment for 7 weeks and a successful pregnancy and healthy baby after ceasing testosterone treatment for 2 months prior (Hassan et al. 2022). Further research in this area will provide important insights for best practice.

Similarly, the research being led by Associate Professor Ken Pang, a researcher at the Murdoch Children’s Research Institute, the Royal Children’s Hospital, and the University of Melbourne, focuses on challenging the assumptions of the reproductive health of trans women. In recent years, paediatric referrals for specialist gender-affirming medical care have increased. For individuals that do access gender-affirming medical care, there are some treatments that impair reproductive function, including GAHT for trans women. While GAHT promotes feminisation, it impairs spermatogenesis (Jindarak et al. 2018; Jiang et al. 2019; Vereecke et al. 2021). This necessitates access to fertility counselling and preservation options, particularly if there is a desire to be a genetic parent in the future. Data from North American studies indicated that rates of fertility preservation were low in trans adolescents (<10%) but the underlying reasons are complex and multifaceted, including the desire for genetically-related children, cost, experiences of dysphoria, and further delays to GAHT (Chen et al. 2017; Nahata et al. 2017). Associate Professor Pang presented data challenging the assumption that trans adolescents assigned male at birth are not interested in sperm cryopreservation. In a study where barriers to accessing fertility preservation (e.g. time to treatment, cost) were reduced, 62% of patients assigned male at birth accessed fertility preservation (Pang et al. 2020). To date, multiple studies indicate that most trans women undergoing GAHT show impaired spermatogenesis (Jindarak et al. 2018; Jiang et al. 2019; Vereecke et al. 2021). However, research by Associate Professor Pang challenged another common assumption that GAHT-induced inhibition of spermatogenesis causes permanent infertility. In a longitudinal study, semen of nine trans women who temporarily stopped GAHT for immediate conception or future reproductive purposes was analysed. Of these patients, six presented with sperm in the initial semen analysis after cessation of GAHT and two had sperm detected in semen analysis within a year of GAHT cessation, while the remaining patient underwent testicular biopsy to recover mature spermatozoa (de Nie et al. 2023). Although preliminary, these findings challenge widely held assumptions about the permanence of infertility secondary to GAHT in trans women. In addition to challenging these prevalent assumptions regarding the reproductive health of trans women, Associate Professor Pang highlighted the importance of counselling around fertility preservation for trans individuals who are considering gender-affirming medical care.

Research from Aotearoa, New Zealand, indicates that both Pacific peoples and Rainbow+ communities experience poorer health outcomes. There is, however, an absence of research about the health and wellbeing of Pacific Rainbow+ communities (LGBTTQIA+ MVPFAFF+), who are at the intersection of these experiences of marginalisation (Thomsen et al. 2021). Seuta’afili Dr Patrick Thomsen (Waipapa Taumata Rau, University of Auckland) presented on key lessons from the recently completed first phase of the Manalagi Project (https://www.manalagi.org/). The name ‘Manalagi’ is a reference to the saying ‘mana-mai-le-lagi’, which refers to the ‘unquestionable legitimacy and spiritual authority gifted by the heavens that is inherent to all’, and positions Pacific Rainbow+ peoples within Pacific cultures and society (Thomsen et al. 2023). Overall, the Manalagi Project aimed to enhance the mana (power) and wellbeing of Pacific Rainbow+ communities in Aotearoa New Zealand through research. In a 3-year study funded by the Health Research Council of New Zealand, the research team collected baseline data pertaining to the health and wellbeing of Pacific Rainbow+ communities. The Pacific Rainbow+ community was integrally involved in the development of the Manalagi survey to ensure it could capture their unique experiences and diverse needs (Thomsen et al. 2023). Community consultation was conducted throughout 2021, comprising 11 co-design sessions across Aotearoa-NZ with Pacific Rainbow+ individuals and their allies. Following extensive community consultation, the HCLC Manalagi Model of Community Engagement was co-designed and comprised of four elements: (1) to honour gafa (genealogies); (2) to connect meaningfully and intentionally with community mana; (3) to listen to understand the aspirations and needs of the community; and (4) to collaborate in the spirit of tautua (service). This allowed the aspirations of participants to be centred in all ongoing work. In implementing the HCLC model, the Manalagi team were able to build trust and legitimacy within Pacific Rainbow+ communities, ensuring that the process was not extractive whilst maintaining the mana of the researchers, who are themselves members of Pacific Rainbow+ communities. Outcomes of this process included the development of a survey tool (Manalagi survey) that was centred on the priorities of community members, including the inclusion of open-text boxes for gender and sexuality questions, and newly co-designed projects. By centering the project in Pacific Rainbow+ communities, cultural knowledge was amplified and contributed to reduced anti-Rainbow+ violence as well as shifting conservative positions within Pacific communities. The Manalagi Survey Community Report (Thomsen et al. 2023), a key output of the first phase, compiled the findings of the survey. The next phases of the Manalagi Project are planned to involve development of a Manalagi television series, a Manalagi Collection at Te Papa Tongarewa (Museum of New Zealand), and a Manalagi stories book. Overall, the Manalagi Project empowered Pacific Rainbow+ communities to co-design this research and as a result created a platform to advance their own projects. Moreover, it represents a step towards acknowledging key issues including societal status and visibility, and how these contribute to the wellbeing of multiply marginalised groups, including Pacific Rainbow+ communities in Aotearoa, New Zealand.

Speakers: Wendy Ingman, Mitchell Lawrence, Shafiq Syed, and Karla Hutt

The symposium on reproductive cancers highlighted advances in the treatment of breast and prostate cancer, novel insights into the processes of endometrial growth, and improvements in fertility preservation during cancer treatments.

In Australia, the incidence of breast cancer is increasing across all age groups, while simultaneously, early detection and improvements in treatment options for patients has lowered the mortality rate (Australian Institute of Health and Welfare 2022). Risk modifier alleles such as BRCA1 and BRCA2 account for ~5% of cases, yet individuals with high mammographic breast density account for ~18% of breast cancer cases (McCormack and dos Santos Silva 2006; Engmann et al. 2017). Alarmingly, approximately 50% of women in first-world countries have this breast cancer risk factor (Sprague et al. 2014). Breast density usually slowly declines with age after 40 years, when screening often begins (Sprague et al. 2014; Kim et al. 2020), although those with persistent breast density maintain a greater risk for breast cancer (Kim et al. 2020). Higher-density breast tissue regions have less abundance of adipose tissue and more stromal collagen, with an altered immune cell composition (Huo et al. 2015). As breast tissue largely develops during puberty, Associate Professor Wendy Ingman (University of Adelaide) and team are investigating pubertal mammary gland development as a determinant of adult breast density (Ghadge et al. 2021). Notably, there is an inverse association between pubertal weight and body mass index (BMI) with adult breast density and breast cancer risk (Andersen et al. 2014; Bertrand et al. 2015; Hopper et al. 2016; Alexeeff et al. 2017; Schoemaker et al. 2017; Pedersen et al. 2022), although protection against breast cancer after a higher childhood BMI remains after adjustment for current breast density (Harris et al. 2011). Dr Ingman and team are investigating the mechanisms behind the protective effect of early life adiposity against breast cancer risk by using a mouse model for hyperphagia-induced adiposity (Alms1^−/−). Their preliminary results suggest that increased adiposity during puberty reduces adult fibroglandular density and delays onset of tumour development. These findings support the notion that increased adiposity during puberty decreases mammographic density and breast cancer risk. However, diet quality is also an important consideration, since a high-fat diet in mice during puberty can increase susceptibility to breast cancer development in adulthood (Aupperlee et al. 2015). Nonetheless, this research suggests that puberty might be a window of opportunity for intervention against breast cancer, whereby good nutrition and a balanced diet could reduce the adult breast cancer risk.

Prostate cancer is the most common cancer in males in 112 countries (Wang et al. 2022) and the second-leading cause of cancer death in men (Sung et al. 2021). Dr Mitchell Lawrence (Monash University) is developing new patient-derived models of prostate cancer, which currently represent only 0.17% of xenografts in international collections (Lawrence et al. 2023). The most commonly used cell lines today were established in the 1970s (Stone et al. 1978; Kaighn et al. 1979; Horoszewicz et al. 1980) and do not reflect clinical heterogeneity. Dr Lawrence and team engraft human tumours into mice to create patient-derived xenograft (PDX) models, enabling tumour biology and therapies to be tested in a manner that addresses patient diversity (Lawrence et al. 2023). The Melbourne Urological Research Alliance (MURAL) has a growing collection of 76 PDXs from 38 patients (Risbridger et al. 2021). While androgen deprivation therapy is one current treatment for advanced prostate cancer, it is not curative and can lead to treatment resistance. Patients on sustained treatment also experience reduced quality of life due to a myriad of side-effects including hot flushes, breast tenderness, fatigue, musculoskeletal complaints, and decreased sexual function and libido. Notably, prostate cancer has a biphasic response to testosterone therapy whereby very low or high concentrations are antiproliferative against tumours. This observation has led to the development of a new therapy to treat advance prostate cancer known as bipolar androgen therapy (BAT) (Schweizer et al. 2016; Teply et al. 2018; Denmeade et al. 2021), which may reduce side-effects compared with sustained use of androgen receptor signalling inhibitors. Clinical trials show a ~30% response rate in reduction of tumour burden with BAT, with varying effectiveness depending on type of tumour (Schweizer et al. 2016; Teply et al. 2018; Denmeade et al. 2021). PDXs are useful tools to study which tumours are likely to respond to BAT – tumour samples include those resistant to current treatments through various mechanisms, and from different metastatic sites. Using castrated mice to represent androgen receptor antagonist therapies, PDXs are classified as ‘resistant’ or ‘responsive’ to BAT by assessing responses following administration of testosterone cypionate to achieve supraphysiological testosterone levels. Results suggest that BAT is more effective when tumours have robust ligand binding due to both high levels of androgen receptor and low levels of ligand-independent androgen receptor variants (James Cuffe, unpubl. data). Future directions of this work include testing existing and new therapy combinations to increase therapy response rates.

Dr Shafiq Syed (University of Newcastle) and his team study endometrial stem cells in reproductive diseases and ageing. Endometrial regeneration occurs at a rate of ~4–10 mm per month (Syed et al. 2020). Excessive regeneration can cause extreme or ectopic growth, while insufficient regeneration can cause uterine adhesions and infertility. Using Pax8-targeted genetic labelling to perform epithelial cell fate tracing in mice, Dr Syed and team discovered that the endometrial epithelium renews itself throughout life, with cells dividing approximately every 3 days (Syed et al. 2020). The team also tested for clonal expansion using random labelling of epithelial cells, where analysis of labelling revealed a pattern consistent with stem cell involvement (Syed et al. 2020). Serial thymidine analogue labelling suggested a stem cell compartment in the glandular tips of the endometrium, which were positive for the classical Wnt reporter gene Axin2 (Syed et al. 2020). Further studies using Axin2 stem cell-derived organoids and multiple mouse models of human menstruation uncovered that AXIN2 ablation compromises endometrial regeneration and that upon oncogenic transformation, AXIN2 presence marked the origin cell for endometrial cancer. Strikingly, Axin2-expressing cells alone gave rise to cancer when mutated. Ongoing work is addressing the effects of uterine ageing on stem cell regeneration and cancer development, and is investigating mechanisms of scarless cellular regeneration in injury repair.

Chemotherapy and radiotherapy damage the ovaries and can cause premature menopause, which increases the risk for glaucoma, cardiovascular disease, dementia, and fertility concerns. Some patients opt for oocyte or embryo cryopreservation, although these do not prevent premature ovarian failure. For pre-pubertal patients, the only option is ovarian tissue cryopreservation and grafting, which can re-introduce cancer. Dr Karla Hutt (Monash University) is developing innovative pharmacological ovarian protectants to preserve fertility during cancer treatments. This involves defining the impact of cancer treatments on ovarian function and fertility, then identifying causal pathways that trigger follicle depletion. Dr. Hutt and team have summarised the literature on ovarian cell death pathway regulators, including apoptotic and non-apoptotic pathways, and their functions throughout life (Stringer et al. 2023). Their latest work is driven by patient requests for information. Reproductive endpoints are rarely reported in clinical trials for cancer treatments and studies do not routinely assess ovarian toxicity, despite the devastating impact of infertility (Cui et al. 2023). To begin to bridge this gap, Dr Hutt’s team have tested a range of treatments, including PARP inhibitors and immunotherapies such as monoclonal antibodies administered to ‘turn off’ components of the immune system. These immune checkpoint inhibitors target key pathway mediators such as programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and cytotoxic T-lymphocyte associated protein 4 (CTLA4). Such therapies are now standard of care for numerous cancers such as melanoma, triple-negative breast cancer, and lung cancer that occur in reproductive age women. Dr Hutt and team found that tumour-free mice administered immune checkpoint inhibitors exhibit a reduction in primordial follicles, impaired ovulation, more oocyte death, and characteristics consistent with endocrine dysfunction. This was associated with an increase in ovarian T cells. Further investigations using the Rag2^−/− mouse model of immune depletion revealed that the ovarian dysfunction observed was in fact dependent on the presence of immune cells. This work importantly highlights that newer immunotherapies may not be better for fertility than traditional cancer treatments. There is thus a pressing need for human clinical data on reproductive outcomes to enable patients to make informed decisions about their reproductive and oncological health. In the absence of such data, several healthcare companies now share preclinical data directly with patients, enabling them to make more informed treatment decisions.

This symposium session highlighted fantastic advances in our understanding of basic biological mechanisms of cancer, which will ultimately contribute to a reduced societal cancer burden. It is highly encouraging that patient-driven advances have been made in Australian reproductive cancer studies, since patient treatment options or access to information has historically been very limited.

Speakers: Craig Smith, Lisa Schwanz, Frank Grutzner, and Michael Garratt

Sexual reproduction is a key step of life allowing for the generation of offspring that carry genetic information from individuals of different sexes. To maximise reproductive success and offspring fitness, species have evolved unique reproductive mechanisms to adapt to the changing environment. For instance, sex determination and sexual development are susceptible to temperature, particularly in non-mammalian species. Despite the importance of reproduction, emerging evidence suggests that mating as an event in the life-history of an organism has a major impact on its lifespan (Garratt et al. 2020). Focusing on non-human species, this symposium session highlighted the evolution of sex chromosomes, the influence of environmental factors on sexual development, and the close association between species life-history and survival.

One long-standing focus of research in reproductive biology has been to define the mechanism of sex determination. Historically, this was perceived as a two-step process, whereby sex chromosomes first direct the embryonic gonads to form ovaries or testes, then gonad-secreted sex hormones feminise or masculinise the rest of the body. Yet not all findings agree with this paradigm, with emerging evidence suggesting parallel sexually dimorphic processes. Associate Professor Craig Smith (Monash University) presented the remarkable story of Gerri, a naturally occurring gynandromorphic chicken that was chromosomally ZZ (male) on one side of the body and largely ZZ on the other, but with 10% ZW (female) cells (Morris et al. 2018). Despite high circulating testosterone level, and low levels of ZW cells, one half of the bird was phenotypically female. The ‘male side’ gonad was a testis, with Sertoli cells, germ cells, and active spermatogenesis, while the ‘female side’ gonad contained both follicles and seminiferous tubules. This example illustrates that sexually dimorphic structures must be at least partly independent of sex steroid effects, adding to evidence that there is a degree of cell-autonomous sexual determination in birds (Zhao et al. 2010). To explain the mysterious process of sex determination in birds, the conserved Z-linked gene, doublesex, and mab-3-related transcription factor 1 (DMRT1) was further investigated (Smith et al. 2009). DMRT1 knock-down in vivo led to feminisation of the embryonic gonads in genetically male (ZZ) embryos, with affected males showing partial sex reversal, suggesting the significance of DMRT1 in avian sex determination. In addition to DMRT1, oestrogen production is also a key factor in primary sex determination in chickens, which is linked to Dmrt1 expression (Ioannidis et al. 2021). Beyond the gonads, however, other combinations of Z-linked genes intertwined with sex steroid hormones, may govern sexual development and secondary characteristics, along with a possible role for post-transcriptional mechanisms. With the advent of modern sequencing technologies and gene editing approaches, future studies will yield new insights into the remarkable phenomenon of cell-autonomous sexual development in birds, and the evolution of diverse mechanisms of sex determination.

In many organisms, the commitment to male or female sex depends on genetics but is sensitive to environmental conditions. Associate Professor Lisa Schwanz (University of New South Wales) is investigating how environmental lability in sexual development can drive evolutionary transitions in sex-determining mechanisms, from genotypic to environmental sex determination (Schwanz et al. 2013; Schwanz et al. 2020). In some reptiles, temperature interacts with genotype to reverse sex, where a temperature-dependent sex determining system can override chromosomal sex. The frequency and fitness of sex-reversed individuals can, in turn, have profound behavioural and fitness consequences on the ecological trajectory of a population. For instance, the Australian Central Bearded Dragon (Pogona vitticeps) exhibits sex reversal, such that high incubation temperatures cause reversal of ZZ genotype embryos (normally male) to female phenotypes (Quinn et al. 2007). Subsequent matings between ZZ females (ZZf) and ZZ males (ZZm) produce only chromosomal male offspring when incubated at intermediate incubation temperatures, while females arise only from temperature-induced sex reversal (ZZf) (Holleley et al. 2015). In laboratory settings, sex-reversed females are larger, more fecund, bolder, and have higher activity than their ZZm or concordant (ZWf) female counterparts. In contrast, although ZZf animals were larger than ZWf animals in wild populations, they did not display the greater reproductive output and body condition seen in captive ZZf juvenile animals (Wild et al. 2022). While differences in fitness-related traits and movement were strongest between reproductive phenotypes rather than sex chromosomes, further research is required to determine the long-term consequences of this observation in the wild. These findings demonstrate that behavioural adaptations may serve as a potential buffering mechanism against transitions to temperature-dependent sex determination under environmental changes, such as climate warming or habitat fragmentation.

Surprisingly, differences in mechanisms of sex determination are evident even between mammalian species. In mammals, sex chromosomes are denoted by X and Y, many with the combination of XX and XY for females and males, respectively. Speciation has led to the independent evolution of sex chromosomes between different mammalian species such as monotremes and therian mammals, with the monotreme sex determination system having five pairs of sex chromosomes that share no homology with the therian sex chromosomes (Rens et al. 2007; Veyrunes et al. 2008). Professor Frank Grutzner (University of Adelaide) has used monotremes to understand the fundamental aspects of the evolution and regulation of sex chromosomes in mammals. In a major finding, Professor Grutzner’s team showed that the platypus sex chromosome lacks many of the epigenetic hallmarks of meiotic sex chromosome inactivation (MSCI) and differential cohesion accumulation (Daish et al. 2015; Murat et al. 2023). They also found that the process of meiotic silencing of unpaired chromatin is common to all mammals (Casey et al. 2017; Murat et al. 2023). The team is currently investigating if platypus chromosome 6, which shares extensive homology to the therian X chromosomes, can provide information about the sex chromosome system in early mammals. These findings collectively highlight that monotremes have been instrumental for comparative genomics research and can provide unique insights into mammalian sex chromosome evolution.

Although reproduction is a crucial aspect of life for offspring production and population maintenance, there is a trade-off between reproduction and lifespan, both of which constrain survival. In fact, sterilisation may increase lifespan, as suggested in a population of castrated sheep (Sugrue et al. 2021). To further understand this phenomenon, Associate Professor Michael Garratt (University of Otago) utilised data collected from zoo housed animals and showed that sterilisation in both males and females through surgical castration and hormonal contraceptive methods, respectively, is associated with increased lifespan. This phenomenon was evident in various mammalian species, including primates, carnivores, and bats. Furthermore, a meta-analysis of published data revealed a similar increase in survival metrics in males after castration and in females treated with contraceptives in both a laboratory setting and in the wild. However, one question left unanswered is why females have a higher life expectancy than males on average in mammalian species (Lemaître et al. 2020). Castration does not completely negate the sex-specific differences in ageing, suggesting the costs of reproduction only in part explain these differences and that there are other contributing factors, including early life sex hormone exposure and environmental conditions (Jiang et al. 2023). Together, these findings shed light on the trade-offs between reproduction and longevity across vertebrates, and how such trade-offs can be altered by means like sterilisation and environmental factors.

To conclude, this symposium explored diverse aspects of sexual reproduction, encompassing evolutionary adaptations, lifespan dynamics, and the intricate mechanisms underlying sex determination. Investigations into sex chromosome evolution, the impact of sterilisation on lifespan extension, and the intriguing phenomena of sex determination in birds, reptiles, and monotremes, have generated cutting-edge knowledge and profound insights. Collectively, these findings highlight the complex interplay between genetic and environmental factors in shaping reproductive strategies and life-history traits across species. Further exploration of these areas will continue to unravel the mechanisms of sexual reproduction and their implications for species survival in the face of environmental change.