PURPOSE:This study estimates the need of IVF/ICSI in Australia as compared to its actual uptake. METHODS:We created a model estimating for the annual demand for IVF/ICSI in a hypothetical infertile population, using demographic data from medical literature and Australian government databases. For each category of infertility (tubal, severe male, endometriosis, anovulation and unexplained), our estimated need for IVF/ICSI was compared to the actual IVF/ICSI uptake (ANZARD 2019). The model consisted of three categories depending on couples' cause of infertility, i.e. couples with absolute indications for IVF/ICSI (couples with severe male factor infertility and tubal obstruction); couples with anovulatory infertility (couples with ovulation disorders) and couples with ovulatory infertility (couples suffering from unexplained infertility and endometriosis). The model was applied to each of these categories to determine the number of couples that would require IVF/ICSI treatment after failing to conceive naturally or after following alternative treatment plans. The main outcomes of this study were the estimate of IVF/ICSI cycles and the difference between the estimate and the reported number of IVF/ICSI cycles (2019 ANZARD report). RESULTS:We estimated that approximately 35,300 couples required IVF/ICSI treatment in Australia in 2019, while in 2019 according to ANZARD, 46,000 couples underwent IVF/ICSI. A higher uptake of IVF/ICSI cycles than expected was specifically reported in couples with unexplained infertility, ovulation disorders and endometriosis, while for tubal and severe male infertility uptake seemed adequate. CONCLUSION:In Australia, there seems to be overservicing of IVF/ICSI, specifically for unexplained, ovulatory and endometriosis-related infertility.

Reliable techniques and criteria for identifying sex and age class are important to understand the population biology of species in the course of wildlife research and monitoring. In platypuses, males have a keratinous spur on the inner surface of the ankle on each hind leg that undergoes characteristic age-related morphological changes within the first 3 years of life. Females, in contrast, do not develop a spur but instead develop a vestigial spur sheath that is lost toward the end of their first year. Spur developmental stages can be used to assign male platypuses to three age categories: ‘juveniles’ (≤12 months old), ‘sub-adults’ (13–24 months old) and ‘adults’ (>2 years of age), which are capable of breeding. Male spurs grow from a mean length of 12.4 (±0.5) mm after emergence from the nesting burrow to 18.6 (±0.6) mm by the end of the first year of life in Victoria. Platypus age categories have at times been inaccurately assigned. Here, we present an annotated pictorial guide of these age categories, suitable for use by wildlife rescuers and researchers in a field setting, and in veterinary surgeries, to facilitate accurate identification by those not familiar with this aspect of platypus biology.

Affiliation: #Authors equally contributed to this work as last authors 1Department of Obstetrics and Gynaecology, Monash University, Melbourne, Victoria, 3168, Australia 2Department of Physiology, Monash University, Melbourne, Victoria, 3168, Australia 3Hudson Institute of Medical Research, Melbourne, Victoria, 3168, Australia 4Melbourne Institute of Plastic Surgery, Malvern, Victoria 3144, Australia

Abstract Background The Egyptian spiny mouse (Acomys cahirinus) is the only known rodent to exhibit true, human-like menstruation and postpartum ovulation, and is an important new model for reproductive studies. Spiny mice do not produce a visible copulatory plug, and calculation of gestational age is therefore restricted by the need to use mated postpartum dams. The current inefficient method of monitoring until parturition to provide a subsequent estimate of gestational age increases study duration and costs. This study addressed this issue by comparing the mating behaviour of spiny mice across the menstrual cycle and proposes a more accurate method for staging and pairing animals that provides reliable estimates of gestational age. In experiment 1, mating behaviour was recorded overnight to collect data on mounting, intromission, and ejaculation (n = 5 pairs per stage) in spiny mice paired at menses and at early and late follicular and luteal phases of the menstrual cycle. In experiment 2, female spiny mice were paired at the follicular or luteal phases of the menstrual cycle to determine any effect on the pairing-birth interval (n = 10 pairs). Results We report a broad mating window of ~ 3 days during the follicular phase and early luteal phase of spiny mice. Males displayed a discrete ‘foot twitch’ behaviour during intromission and a brief copulatory lock during ejaculation. Litters were delivered after 40–43 days if pairing occurred during the mating window, compared with 46–48 days for spiny mice paired in the late luteal phase. When pairing occurred during the late luteal phase or menses no mating activity was observed during the recording period. Conclusion This study clearly demonstrates an effect of the menstrual cycle on mating behaviour and pregnancy in the spiny mouse and provides a reliable and more effective protocol for estimating gestational age without the need for postpartum dams.

Although certain organisms are chosen and employed to better understand a specific problem in biology (so-called model organisms), sometimes an animal model reveals its' biomedical importance by happenstance. In many ways, the advent of spiny mice (Acomys) as an emerging model to study regeneration and menstruation stands as a case study in scientific pseudoserendipity (Diaz de Chumaceiro, 1995). As we recount in this chapter, the discovery of these phenotypes, while not entirely accidental, was nonetheless unexpected. In addition to recounting how we uncovered these unusual mammalian traits, we outline recent work by our groups and others that has begun to outline the cellular and genetic mechanisms underlying bonafide mammalian tissue regeneration and a human-like mode of reproduction in spiny mice.

Freshwater fish populations are declining with many small, Australian fish species at risk of extinction within the next twenty-years. Cryopreservation of reproductive cells and tissues makes it possible to reproduce individuals from a species even after they are extinct in the wild. We describe the successful cryopreservation of ovarian tissue in the Murray River Rainbowfish, Melanotaenia fluviatilis (Order: Atheriniformes). Histology showed that oogonia are 13.70 µm ± 1.75 µm in size, stain positive for germ-line marker Vasa, and represent approximately 2.29 ± 0.81% of cells in the ovary. Flow cytometry was used to analyse ovarian cell suspensions, requiring an optimised tissue digestion protocol. We found that 0.25% trypsin with 1.13 mM EDTA produced cell suspensions with the highest viability (76.28 ± 4.64%) and the highest number of cells recovered per gram of tissue (1.2 × 108 ± 4.4 × 107 cells/g). Subsequent sorting of ovarian cell suspensions by flow cytometry increased oogonial cells in suspension from 2.53 ± 1.31% in an unsorted sample to 5.85 ± 4.01% in a sorted sample (p = 0.0346). Cryopreservation of ovarian tissue showed DMSO-treated samples had higher cell viability post-thaw (63.5 ± 18.2%) which was comparable to fresh samples (82.5 ± 7.1%; p = 0.36). Tissue cryopreserved in 2.0 M DMSO had the highest cell viability overall (76.07 ± 3.89%). This protocol could be applied to bio-banking programs for other species in the Melanotaeniidae, and perhaps species in other families and orders of Australian fish.

Menstruation, the cyclical breakdown of the uterine lining, is arguably one of evolution's most mysterious reproductive strategies. The complexity and rarity of menstruation within the animal kingdom is undoubtedly a leading contributor to our current lack of understanding about menstrual function and disorders. In particular, the molecular and environmental mechanisms that drive menstrual and fertility dysregulation remain ambiguous, owing to the restricted opportunities to study menstruation and model menstrual disorders in species outside the primates. The recent discovery of naturally occurring menstruation in the Egyptian spiny mouse (Acomys cahirinus) offers a new laboratory model with significant benefits for prospective research in women's health. This review summarises current knowledge of spiny mouse menstruation, with an emphasis on spiral artery formation, inflammation and endocrinology. We offer a new perspective on cycle variation in menstrual bleeding between individual animals, and propose that this is indicative of fertility success. We discuss how we can harness our knowledge of the unique physiology of the spiny mouse to better understand vascular remodelling and its implications for successful implantation, placentation, and foetal development. Our research suggests that the spiny mouse has the potential as a translational research model to bridge the gap between bench to bedside and provide improved reproductive health outcomes for women.

Egyptian spiny mice are the only known species to have human-like menstruation and a postpartum ovulation. Unfortunately, no endocrine or morphological evidence has been provided for a postpartum ovulation in spiny mice, and while later stages of pregnancy have been well studied, early events including embryo implantation and spiral artery remodelling have not been reported. This study compared the sex steroid endocrinology and reproductive tract morphology of dams at eight timepoints (n = 40) postpartum to determine the timing of ovulation and the timing and invasiveness of embryo implantation in A. cahirinus . Reproductive tracts were fixed and stained for histology and immunohistochemistry, and plasma was prepared for enzyme-linked immunosorbent assay. Ovarian histology and estradiol-17B concentrations indicate ovulation within 48 h of parturition and then immediate resumption of follicular growth. Uterine histology and immunohistochemistry revealed progressive epithelial repair, endometrial growth and spiral artery assembly and remodelling in dams postpartum. Blastocysts were seen in the uterine lumen at day 4–5 postpartum and embryos had implanted superficially with minimal stromal invasion by day 5–6. This study provides further evidence for the unique, humanesque reproductive biology of spiny mice and for a postpartum ovulation using endocrine and morphological changes observed during early pregnancy. Taken together, our data suggest that spiny mice may act as appropriate models of human pregnancy disorders such as implantation failure or pre-eclampsia.

STUDY QUESTION: Does the naturally menstruating spiny mouse go through menopause? SUMMARY ANSWER: Our study is the first to show a natural and gradual menopausal transition in a rodent. WHAT IS KNOWN ALREADY: Age-related depletion of the human ovarian reserve (OvR) leads to menopause, the permanent cessation of menstruation and reproduction. Current rodent models of menopause are inappropriate for inferences of the human condition, as reproductive senescence is abrupt or induced through ovariectomy. The spiny mouse is the only confirmed rodent with a naturally occurring menstrual cycle. STUDY DESIGN, SIZE, DURATION: Histological assessment of virgin spiny mice occurred in females aged 6 months (n = 14), 1 year (n = 7), 2years (n = 13), 3 years (n=9) and 4 years (n=9). Endocrinology was assessed in a further 9 females per age group. Five animals per group were used for ovarian stereology with additional ovaries collected at prenatal Day 35 (n = 3), day of birth (n = 5), postnatal Days 35 (n = 5) and 100 (n = 5) and 15 months (n = 5). PARTICIPANTS/MATERIALS, SETTING, METHODS: Morphological changes in the reproductive system were examined using hematoxylin and eosin stains. Proliferating cell nuclear antigen immunohistochemistry assessed endometrial proliferation and sex steroids estradiol and testosterone were assayed using commercial ELISA kits. MAIN RESULTS AND THE ROLE OF CHANCE: The proportion of females actively cycling was 86% at 6 months, 71% at 1 year, 69% at 2 years, 56% at 3 years and 44% at 4 years. Uterine and ovarian weights declined steadily from 1 year in all groups and corresponded with loss of uterine proliferation (P<0.01). Estradiol was significantly decreased at 1 and 2 years compared to 6-month-old females, before becoming erratic at 3 and 4 years, with no changes in testosterone across any age. Fully formed primordial follicles were observed in prenatal ovaries. Aging impacted on both OvR and growing follicle numbers (P<0.001-0.0001). After the age of 3years, the follicle decline rate increased more than 5-fold. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study in a novel research rodent whereby reagents validated for use in the spiny mouse were limited. WIDER IMPLICATIONS OF THE FINDINGS: The gradual, rather than sudden, menopausal transition suggests that the spiny mouse is a more appropriate perimenopausal model than the current rodent models in which to examine the neuroendocrine pathways that encompass all hormonal interactions in the hypothalamic-pituitary-gonadal axis. The logistic, ethical and economic advantages of such a model may reduce our reliance on primates in menopause research and enable more thorough and invasive investigation than is possible in humans.

Professor Short was a great scholar, researcher, teacher, mentor and raconteur whose research spanned 60 years and an incredible breadth of topics.

The menstruating spiny mouse is the first rodent identified to exhibit natural spontaneous decidualisation, cyclical endometrial shedding and regeneration. While the spiny mouse shares several primate-like characteristics in its reproductive biology, it has not been established whether pseudopregnancy can be induced or if its cycles can be synchronised as in non-human mammals. Here we describe attempts to induce pseudopregnancy and synchronisation of menstrual cycles (i.e. Whitten effect) in spiny mice. Virgin females (n=3-8 per group) underwent one of the following procedures to induce pseudopregnancy: daily vaginal lavage only (control), progesterone injection, mechanical stimulation of the cervix and sterile mating. A separate cohort was also exposed to male-soiled bedding to assess the Whitten effect. Pseudopregnancy was deemed successful if females presented with extended (>12 consecutive days) leukocytic vaginal cytology. No female from any method of induction met this criterion. In addition, the menstrual cycles of a group of six females could not be synchronised, nor immediate ovulation induced via exposure to male-soiled bedding. These responses indicate that the spiny mouse does not behave as a typical rodent. Like higher-order primates, the spiny mouse exhibits a relatively rare reproductive strategy, of failure to show pseudopregnancy or cyclical synchronisation. This is further endorsement of the use of this species as a versatile animal model for translational studies of menstruation and fertility.

Maternal care in platypuses has never been rigorously studied due to the difficulty in locating and accessing nesting burrows. Here we describe the maternal behaviour of a captive female platypus and the growth and development of her offspring over 11 breeding seasons. We located a nesting burrow and inserted a camera to record the activity and behaviour of the female and her offspring. We also measured the female’s food intake during lactation. The ages of the offspring were assigned to developmental milestones including opening of the eyes and development of pelage. Twins (n=4) were left alone for periods longer than 24h by the mother at an earlier age than single nestlings (n=2). The dietary energy intake of the breeding female was more than double that of non-lactating females in the last month of lactation, indicating the large energy requirements of milk production. The mean age of young at emergence from the burrow was 128±1 days and in 60% of nestlings emergence occurred after weaning. This suggests a rapid transition from a completely milk-based diet to a diet of aquatic macroinvertebrates. The techniques we developed have allowed us to study maternal care in platypuses and the development of nestlings, both of which were previously only poorly understood.

The menstruating Egyptian spiny mouse has recently been proposed as a new animal model for reproductive health research. Unfortunately, little is known about reproduction in males. This study compared several characteristics of sperm function before and after cryopreservation. Epididymal spermatozoa were cryopreserved in different concentrations of raffinose and skim milk and tested for motility and membrane integrity (Experiment 1). Further evaluations of motility, plasma membrane and acrosome integrity, mitochondrial membrane potential and DNA integrity were conducted with the addition of l-glutamine to the extender (Experiment 2). The results show that, following cryopreservation, motility and membrane integrity were reduced, but were better maintained in the presence of l-glutamine (P<0.05). Moreover, although all sperm parameters were significantly reduced following cryopreservation (P<0.05), most cryopreserved spermatozoa retained acrosome, membrane and DNA integrity while also maintaining motility and mitochondrial membrane potential. This study provides a new step towards the development of assisted reproductive techniques and archiving the important genetics of the world’s only known menstruating rodent.

Oocyte quality and reproductive outcome are negatively affected by advanced maternal age, ovarian stimulation and method of oocyte maturation during assisted reproduction; however, the mechanisms responsible for these associations are not fully understood. The aim of this study was to compare the effects of ageing, ovarian stimulation and in-vitro maturation on the relative levels of transcript abundance of genes associated with DNA repair during the transition of germinal vesicle (GV) to metaphase II (MII) stages of oocyte development. The relative levels of transcript abundance of 90 DNA repair-associated genes was compared in GV-stage and MII-stage oocytes from unstimulated and hormone-stimulated ovaries from young (5–8-week-old) and old (42–45-week-old) C57BL6 mice. Ovarian stimulation was conducted using pregnant mare serum gonadotropin (PMSG) or anti-inhibin serum (AIS). DNA damage response was quantified by immunolabeling of the phosphorylated histone variant H2AX (γH2AX). The relative transcript abundance in DNA repair genes was significantly lower in MII oocytes compared to GV oocytes in young unstimulated and PMSG stimulated but was higher in AIS-stimulated mice. Interestingly, an increase in the relative level of transcript abundance of DNA repair genes was observed in MII oocytes from older mice in unstimulated, PMSG-stimulated and AIS-stimulated mice. Decreased γH2AX levels were found in both GV oocytes (82.9%) and MII oocytes (37.5%) during ageing in both ovarian stimulation types used (PMSG/AIS; p < 0.05). In conclusion, DNA repair relative levels of transcript abundance are altered by maternal age and the method of ovarian stimulation during the GV-MII transition in oocytes.

The Egyptian or Common spiny mouse (A. cahirinus) is the first rodent species to show human-like menstruation and spontaneous decidualisation. We consider from these, and its other, human-like characteristics that this species will be a more useful and appropriate small animal model for human reproductive studies. Based on this, there is a need to develop specific laboratory-based assisted reproduction protocols including superovulation, in-vitro fertilisation, embryo cryopreservation and transfer to expand and make this model more relevant. Because standard rodent superovulation has not been successful in the spiny mouse, we have selected to test a human protocol. Female spiny mice will receive a subcutaneous GnRH agonist implant and be allowed to recover. Menstrual cycle lengths will then be allowed to stabilize prior to ovarian stimulation. After recovery, females will be injected IP once a day for 4 days with a FSH analogue, to induce follicular growth, and on day 5 will be injected IP with a hCG analogue to trigger ovulation. Females will either be culled 36hrs after trigger to collect oocytes or immediately paired with a stud male and two cell embryos collected 48hrs later. Mature oocytes will be inseminated using fresh spiny mouse spermatozoa and all in-vitro grown and in-vivo collected two cell embryos will be cryopreserved using methods developed in a close spiny mouse relative, the Mongolian gerbil. For embryo transfer, vitrified embryos will be rapidly warmed and non-surgically transferred to surrogate mice. Surrogates will be monitored until pregnancy is apparent (roughly 30 days) and then left undisturbed until birth, 38–40 days after transfer. By successfully developing robust assisted reproduction protocols in A. cahirinus we will be able to use this rodent as a more effective model for human reproduction.

Fish populations continue to decline globally, signalling the need for new initiatives to conserve endangered species. Over the past two decades, with advances in our understanding of fish germ line biology, new exsitu management strategies for fish genetics and reproduction have focused on the use of germ line cells. The development of germ cell transplantation techniques for the purposes of propagating fish species, most commonly farmed species such as salmonids, has been gaining interest among conservation scientists as a means of regenerating endangered species. Previously, exsitu conservation methods in fish have been restricted to the cryopreservation of gametes or maintaining captive breeding colonies, both of which face significant challenges that have restricted their widespread implementation. However, advances in germ cell transplantation techniques have made its application in endangered species tangible. Using this approach, it is possible to preserve the genetics of fish species at any stage in their reproductive cycle regardless of sexual maturity or the limitations of brief annual spawning periods. Combining cryopreservation and germ cell transplantation will greatly expand our ability to preserve functional genetic samples from threatened species, to secure fish biodiversity and to produce new individuals to enhance or restore native populations.

Globally, fish populations are in decline from overfishing, habitat destruction and poor water quality. Recent mass fish deaths in Australia’s Murray–Darling Basin highlight the need for improved conservation methods for endangered fish species. Cryopreservation of testicular tissue allows storage of early sperm precursor cells for use in generating new individuals via surrogacy. We describe successful isolation and cryopreservation of spermatogonia in an Australian rainbowfish. Testis histology showed rainbowfish spermatogonia are large (> 10 μm) and stain positive for Vasa, an early germ line-specific protein. Using size-based flow cytometry, testis cell suspensions were sorted through “A” (> 9 μm) and “B” gates (2–5 μm); the A gate produced significantly more Vasa-positive cells (45.0% ± 15.2%) than the “B” gate (0.0% ± 0.0%) and an unsorted control (22.9% ± 9.5%, p < 0.0001). The most successful cryoprotectant for “large cell” (> 9 μm) viability (72.6% ± 10.5%) comprised 1.3 M DMSO, 0.1 M trehalose and 1.5% BSA; cell viability was similar to fresh controls (78.8% ± 10.5%) and significantly better than other cryoprotectants ( p < 0.0006). We have developed a protocol to cryopreserve rainbowfish testicular tissue and recover an enriched population of viable spermatogonia. This is the first step in developing a biobank of reproductive tissues for this family, and other Australian fish species, in the Australian Frozen Zoo.

Keloids are benign tumours caused by abnormal wound healing driven by increased expression of cytokines, including activin A. This study compared effects of activins on normal and keloid-derived human dermal fibroblasts and investigated a novel treatment for keloids using follistatin. Normal skin and keloid tissue samples from 11 patients were used to develop primary fibroblast cultures, which were compared in terms of their histology and relevant gene (qRT-PCR and RNAseq) and protein (ELISA) expression. Activin A (INHBA) and connective tissue growth factor (CTGF) gene expression were significantly upregulated in keloid fibroblasts, as was activin A protein expression in cell lysates and culture medium. Activator protein 1 inhibitor (SR11302) significantly decreased INHBA and CTGF expression in keloid fibroblasts and a single treatment of follistatin over 5 days significantly inhibited activin and various matrix-related genes in keloid fibroblasts when compared to controls. Follistatin, by binding activin A, suppressed CTGF expression suggesting a novel therapeutic role in managing keloids and perhaps other fibrotic diseases.

We determined patterns of burrow use by juvenile platypuses (Ornithorhynchus anatinus) in their natal home range, and evaluated associations between burrows and vegetation. Between March 2015 and March 2017, we captured seven juvenile platypuses along a 3-km stretch of Badger Creek, Victoria and fitted them with radiotransmitters. We recorded the locations of animals in their burrows daily while transmitters were attached (range: 14-132 days). Juveniles used 74 different burrows, with each using 11 +/- 2 burrows. Overall, 65% of burrows (48) were used once, 22% (16) were used between 2 and 9 times (moderate-use), and 13% (10) were used frequently (> 10 times). No juveniles dispersed during the monitoring period (14-132 days). Although some association was observed between burrow use and particular vegetation communities, vegetation was not a strong factor driving site selection of burrows. Use of multiple burrows may allow juveniles to avoid competition with conspecifics, reduce exposure to ectoparasites, and develop shelter-seeking behavior. Juvenile platypuses remained in their natal home range, where conditions are likely to be good because they supported recent breeding, while completing their growth and development prior to dispersal.