Fertilisation and larval development in an Antarctic bivalve, Laternula elliptica, under reduced pH and elevated temperatures

Elevated temperatures associated with ocean warming and acidification can influence development and, ultimately, success of larval molluscs. The effect of projected oceanic changes on fertilisation and larval development in an Antarctic bivalve, Laternula elliptica, was investigated through successive larval stages at ambient temperature and pH conditions (-1.6°C and pH 7.98) and conditions representative of projections through to 2100 (-0.5°C to +0.4°C and pH 7.80 to pH 7.65). Where significant effects were detected, increased temperature had a consistently positive influence on larval development, regardless of pH level, while effects of reduced pH varied with larval stage and incubation temperature. Fertilisation was high and largely independent of stressors, with no loss of gamete viability. Mortality was unaffected at all development stages under experimental conditions. Elevated temperatures reduced occurrences of abnormalities in D-larvae and accelerated larval development through late veliger and D-larval stages, with D-larvae occurring 5 d sooner at 0.4°C compared to ambient temperature. Reduced pH did not affect occurrences of abnormalities in larvae, but it slowed the development of calcifying stages. More work is required to investigate the effects of developmental delays of the magnitude seen here in order to better determine the ecological relevance of these changes on longer term larval and juvenile success.

In vitro fertilisation for unexplained subfertility

Peer reviewed

Particle flux characterisation and sedimentation patterns of protistan plankton during the iron fertilisation experiment LOHAFEX in the Southern Ocean

The taxonomic composition and types of particles comprising the downward particle flux were examined during the mesoscale artificial iron fertilisation experiment LOHAFEX. The experiment was conducted in low-silicate waters of the Atlantic Sector of the Southern Ocean during austral summer (January-March 2009), and induced a bloom dominated by small flagellates. Downward particle flux was low throughout the experiment, and not enhanced by addition of iron; neutrally buoyant sediment traps contained mostly faecal pellets and faecal material apparently reprocessed by mesozooplankton. TEP fluxes were low, <5 mg GX eq/m**2/day, and a few phytodetrital aggregates were found in the sediment traps. Only a few per cent of the POC flux was found in the traps consisting of intact protist plankton, although remains of taxa with hard body parts (diatoms, tintinnids, thecate dinoflagellates and foraminifera) were numerous, far more so than intact specimens of these taxa. Nevertheless, many small flagellates and coccoid cells, belonging to the pico- and nanoplankton, were found in the traps, and these small, soft-bodied cells probably contributed the majority of downward POC flux via mesozooplankton grazing and faecal pellet export. TEP likely played an important role by aggregating these small cells, and making them more readily available to mesozooplankton grazers.

Ecological stoichiometry controls the transformation and retention of plant-derived organic matter to humus in response to nitrogen fertilisation

Carbon (C) sequestration in soils is a means for increasing soil organic carbon (SOC) stocks and is a potential tool for climate change mitigation. One recommended management practice to increase SOC stocks is nitrogen (N) fertilisation, however examples of positive, negative or null SOC effects in response to N addition exist. We evaluated the relative importance of plant molecular structure, soil physical properties and soil ecological stoichiometry in explaining the retention of SOC with and without N addition. We tracked the transformation of 13C pulse-labelled buffel grass (Cenchrus ciliaris L.), wheat (Triticum aestivum L.) and lucerne (Medicago sativa L.) material to the <53 μm silt + clay soil organic C fraction, hereafter named “humus”, over 365-days of incubation in four contrasting agricultural soils, with and without urea-N addition. We hypothesised that: a) humus retention would be soil and litter dependent; b) humus retention would be litter independent once litter C:N ratios were standardised with urea-N addition; and c) humus retention would be improved by urea-N addition. Two and three-way factorial analysis of variance indicated that 13C humus was consistently soil and litter dependent, even when litter C:N ratios were standardised, and that the effect of urea-N addition on 13C humus was also soil and litter dependent. A boosted regression analysis of the effect of 44 plant and soil explanatory variables demonstrated that soil biological and chemical properties had the greatest relative influence on 13C humus. Regression tree analyses demonstrated that the greatest gains in 13C humus occurred in soils of relatively low total organic C, dissolved organic C and microbial biomass C (MBC), or with a combination of relatively high MBC and low C:N ratio. The greatest losses in 13C humus occurred in soils with a combination of relatively high MBC and low total N or increasing C:N ratio. We conclude that soil variables involved in soil ecological stoichiometry exert a greater relative influence on incorporating organic matter as humus compared to plant molecular structure and soil physical properties. Furthermore, we conclude that the effect of N fertilisation on humus retention is dependent upon soil ecological stoichiometry.

Regulating IVF and pre-implantation tissue-typing for the creation of "saviour siblings" : a harm analysis

Scientific discoveries, developments in medicine and health issues are the constant focus of media attention and the principles surrounding the creation of so called ‘saviour siblings’ are of no exception. The development in the field of reproductive techniques has provided the ability to genetically analyse embryos created in the laboratory to enable parents to implant selected embryos to create a tissue-matched child who may be able to cure an existing sick child. The research undertaken in this thesis examines the regulatory frameworks overseeing the delivery of assisted reproductive technologies (ART) in Australia and the United Kingdom and considers how those frameworks impact on the accessibility of in vitro fertilisation (IVF) procedures for the creation of ‘saviour siblings’. In some jurisdictions, the accessibility of such techniques is limited by statutory requirements. The limitations and restrictions imposed by the state in relation to the technology are analysed in order to establish whether such restrictions are justified. The analysis is conducted on the basis of a harm framework. The framework seeks to establish whether those affected by the use of the technology (including the child who will be created) are harmed. In order to undertake such evaluation, the concept of harm is considered under the scope of John Stuart Mill’s liberal theory and the Harm Principle is used as a normative tool to judge whether the level of harm that may result, justifies state intervention or restriction with the reproductive decision-making of parents in this context. The harm analysis conducted in this thesis seeks to determine an appropriate regulatory response in relation to the use of pre-implantation tissue-typing for the creation of ‘saviour siblings’. The proposals outlined in the last part of this thesis seek to address the concern that harm may result from the practice of pre-implantation tissue-typing. The current regulatory frameworks in place are also analysed on the basis of the harm framework established in this thesis. The material referred to in this thesis reflects the law and policy in place in Australia and the UK at the time the thesis was submitted for examination (December 2009).

The Commercial Surrogacy Dilemma

In November 2009 the England and Wales High Court (Family Division) granted a parental order pursuant to s30 Human Fertilisation and Embryology Act 1990 in respect of twins who came to be in the custody and control of the applicants (Mr and Mrs A) through a surrogacy arrangement. The particularly unusual and interesting aspect of this case is that, on the evidence, Mr and Mrs A had paid expenses to the surrogate above and beyond those allowed by the legislation, thus creating a commercial surrogacy arrangement. Commercial surrogacy arrangements involve the payment of money to the surrogate mother in excess of those expenses which have been reasonably incurred pursuant to the surrogacy arrangement. This case is relevant to Queensland law because commercial surrogacy arrangements are also prohibited in Queensland and, as in the United Kingdom, the court cannot make a parentage order unless it is satisfied the surrogacy arrangement is not a commercial surrogacy arrangement.

History behind the egg: Women's comprehensive histories and IVF outcomes, Australia, 2008-2009 [abstract]

The individual history of infertile women, as well as their age, may influence their response to in vitro fertilisation (IVF) cycles. This study examined the associations between women’s histories and two IVF outcomes: eggs aspirated (EA) and proportion with normal, two-pronuclei (2PN), fertilisation. This is a cross-sectional survey of infertile women (n=141, 27-46 years) from a multi-centre clinical sample. Participants completed a survey of socio-demographic, relationship, lifestyle, reproductive and fertility factors, medical conditions and recurrent symptoms. Among participants with heterosexual partners (n=122), associations between women’s histories and EA or 2PN fertilisation were analysed using linear and logistic modelling, respectively, adjusted for age at EA and accounting for multiple IVF cycles (n=313 cycles). Participants aged 35+ years had reproductive histories of miscarriage only (16.9%), termination only (9.9%) or birth+termination (5.6%) that were 2-, 3- and 4-fold higher, respectively, than those aged <35 years (7.1%, 2.9%, 1.4%). More years of oral contraceptive use were associated with a lower mean EA: never used, 14.6 EA; 0-2 years, 11.7 EA; 3-5 years, 8.6 EA; 6þ years, 8.2 EA (p=.04). Participants with polycystic ovary syndrome had a higher mean EA (11.5) than those without the condition (8.3 EA, p<.01). Participants in trade or service occupations had lower proportions of 2PN fertilisation (51.7%) than participants in other occupations (professional, 58.6%; manual/other, 63.6%, p<.02). Increasing women’s age and prolonged used of oral contraceptives were associated with lower EA from IVF cycles; PCOS was associated with higher EA. Occupational exposures may have a detrimental effect on normal fertilisation rates.

Early users of fertility treatment with hormones and IVF : women who live in major cities and have private health insurance

Objective: To identify early users (women aged <34 years) of fertility treatment with hormones and in vitro fertilisation (IVF). Methods: A cross-sectional survey of infertile women from fertility clinics (n=59) and from the community (Australian Longitudinal Study on Women's Health participants) who had (n=121) or had not (n=110) used hormones/IVF as treatment for infertility. Associations between socio-demographic, reproductive and lifestyle factors, medical conditions and recurrent symptoms and using treatment (or not) were analysed using multivariable logistic regression. Results: Among infertile women who had used treatment (community vs clinic), women from clinics had lower odds of living outside major cities, using hormones only, i.e., not IVF, or recurrent headaches/migraines, severe tiredness, or stiff/painful joints; and higher odds of recent diagnoses of urinary tract infection or anxiety disorder. Compared to infertile women who had not used treatment, women from clinics had lower odds of living outside major cities, recurrent allergies or severe tiredness; and higher odds of having private health insurance for hospital or ancillary services, recent diagnosis of polycystic ovary syndrome or recurrent constipation. Conclusions: Compared to infertile women in the community, living in major cities and having private health insurance are associated with early use of treatment for infertility at specialist clinics by women aged <34 years. Implications: These results provided evidence of inequity of services for infertile women.

History behind the IVF egg: Are women's comprehensive histories associated with the number of eggs collected, or fertilised normally, from repeated IVF cycles? [abstract]

Most studies of in vitro fertilisation (IVF) outcomes use cycle-based data and fail to account for women who use repeated IVF cycles. The objective of this study was to examine the association between the number of eggs collected (EC) and the percentage fertilised normally, and women’s self-reported medical, personal and social histories. This study involved a crosssectional survey of infertile women (aged 27-46 years) recruited from four privately-owned fertility clinics located in major cities of Australia. Regression modeling was used to estimate the mean EC and mean percentage of eggs fertilised normally: adjusted for age at EC. Appropriate statistical methods were used to take account of repeated IVF cycles by the same women. Among 121 participants who returned the survey and completed 286 IVF cycles, the mean age at EC was 35.2 years (SD 4.5). Women’s age at EC was strongly associated with the number of EC: <30 years, 11.7 EC; 30.0-< 35 years, 10.6 EC; 35.0-<40.0 years, 7.3 EC; 40.0+ years, 8.1 EC; p<.0001. Prolonged use of oral contraceptives was associated with lower numbers of EC: never used, 14.6 EC; 0-2 years, 11.7 EC; 3-5 years, 8.5 EC; 6þ years, 8.2 EC; p=.04. Polycystic ovary syndrome (PCOS) was associated with more EC: have PCOS, 11.5 EC; no, 8.3 EC; p=.01. Occupational exposures may be detrimental to normal fertilisation: professional roles, 58.8%; trade and service roles, 51.8%; manual and other roles, 63.3%; p=.02. In conclusion, women’s age remains the most significant characteristic associated with EC but not the percentage of eggs fertilised normally.

Infertility resolved with or without fertility treatment in Australian women aged 31-36 years: A prospective, population-based study [abstract]

Birth outcomes during a three year period were compared for women with a history of infertility who did or did not use fertility treatment with hormones and/or in vitro fertilisation. Participants in the Australian Longitudinal Study on Women’s Health born in 1973-78 were randomly selected from the universal public health insurance database and completed up to five mailed surveys (1996-2009). Participants reported on their infertility and use of treatment at age 28-33 years (survey 4 (S4) in 2006) and 31-36 years (survey 5 (S5) in 2009). The odds of resolved infertility at S5 were estimated using logistic regression with adjustment for age, area of residence, private health insurance and male infertility. Among 7280 women who responded to both S4 and S5, 18.6% (n=1378) reported infertility. More than half (n=804, 56.8%) of these women did not use treatment and 43.9% (n=347) gave birth between S4 and S5. Compared to infertile women who did not use treatment, women who used treatment were more likely at S5 to have recently given birth (odds ratio (OR) = 1.59, 95% CI 1.26-2.00) or be pregnant (OR = 1.77, 1.27-2.46). Further, women who used treatment were more likely to have twins (3.37, 1.18-9.62), premature births (1.52, 0.95-2.43), or low birthweight babies (1.83, 0.70-2.53) compared to women who gave birth without using treatment. Many women aged up to 36 years with a history of infertility can conceive naturally over a three year period without the use of treatment.Women who have never had a prior birth may need to use treatment to resolve their infertility but they are at higher risk of poorer perinatal outcomes, such as premature or low birthweight babies.

Miscarriage, preterm delivery, and stillbirth: Large variations in rates within a cohort of Australian women

Objectives We aimed to use simple clinical questions to group women and provide their specific rates of miscarriage, preterm delivery, and stillbirth for reference. Further, our purpose was to describe who has experienced particularly low or high rates of each event. Methods Data were collected as part of the Australian Longitudinal Study on Women's Health, a national prospective cohort. Reproductive histories were obtained from 5806 women aged 31–36 years in 2009, who had self-reported an outcome for one or more pregnancy. Age at first birth, number of live births, smoking status, fertility problems, use of in vitro fertilisation (IVF), education and physical activity were the variables that best separated women into groups for calculating the rates of miscarriage, preterm delivery, and stillbirth. Results Women reported 10,247 live births, 2544 miscarriages, 1113 preterm deliveries, and 113 stillbirths. Miscarriage was correlated with stillbirth (r = 0.09, P<0.001). The calculable rate of miscarriage ranged from 11.3 to 86.5 miscarriages per 100 live births. Women who had high rates of miscarriage typically had fewer live births, were more likely to smoke and were more likely to have tried unsuccessfully to conceive for ≥12 months. The highest proportion of live preterm delivery (32.2%) occurred in women who had one live birth, had tried unsuccessfully to conceive for ≥12 months, had used IVF, and had 12 years education or equivalent. Women aged 14–19.99 years at their first birth and reported low physical activity had 38.9 stillbirths per 1000 live births, compared to the lowest rate at 5.5 per 1000 live births. Conclusion Different groups of women experience vastly different rates of each adverse pregnancy event. We have used simple questions and established reference data that will stratify women into low- and high-rate groups, which may be useful in counselling those who have experienced miscarriage, preterm delivery, or stillbirth, plus women with fertility intent.

Fertility and infertility : studies in reproductive epidemiology in Australia

Worldwide, there are few large-scale epidemiological studies on infertility. In Australia, population-based research on infertility is limited to a few small-scale studies. Therefore, the prevalence of infertility and unmet need for specialist medical advice and treatment cannot be estimated reliably. Women who have used assisted reproductive technologies (ART) are recorded in treatment registries. However, there are many infertile women who are excluded from these clinical populations because they neither seek advice nor use treatment. The thesis was based on a biopsychosocial model of health and used the methods of reproductive epidemiology to address the lack of national data on the prevalence of infertility in Australia. Firstly, numbers of births and pregnancy losses were investigated in two generations of women participating in the Australian Longitudinal Study on Women’s Health (ALSWH). The ALSWH is a broad-ranging, longitudinal examination of biological, psychological and social factors that impact on women’s health and wellbeing. Women from three age cohorts were randomly sampled from the population using the universal public health insurance (i.e., Medicare) database and ALSWH participants were representative of the female population. However, the studies in the thesis only involved data from two cohorts. The younger cohort were born in 1973-78 and completed up to four mailed surveys between 1996 (when they were aged 18-23 years, n=14247) and 2006 (28-33 years, n=9145). The mid-aged cohort were born in 1946-51 and completed four mailed surveys between 1996 (when they were aged 45-50 years n=13715) and 2004 (53-58 years, n=10905). Compared to other studies that focus on outcomes of single pregnancies, these studies included all pregnancy outcomes by developing comprehensive reproductive histories for each woman. Pregnancy outcomes included birth, miscarriage, stillbirth, termination and ectopic pregnancy. Women in the youngest cohort (born in 1973-78) were only just reaching their peak childbearing years and many (44%) had yet to report their first pregnancy outcome. Women from the mid-aged cohort (born 1946-51) had completed their reproductive lives and 92% were able to report on their lifetime pregnancy outcomes. Pregnancy losses, especially miscarriage, were common for both generations of women. Secondly, the prevalence of infertility, seeking medical advice and using treatment was identified for these two generations of women. For the older generation, the lifetime prevalence of infertility and demand for treatment was investigated in the context of the specialist medical services which became available circa 1980. By this time, however, most of these older women had already been pregnant and completed their families. For women who experienced infertility (11%), their options for advice and treatment were limited and less than half (42%) had used any treatment. More recently for the younger generation of women, who were aged 28-33 years in 2006, specialist advice and treatment were extensively available. Among women who had tried to conceive or had been pregnant (n=5936), 17% had experienced infertility and the majority (72%) were able to access medical advice. However, after seeking advice only half of these infertile women had used treatment with fertility hormones or in vitro fertilisation (IVF). Overall for infertile women aged up to 33 years, only one-third had used these treatments. Thirdly, the barriers to accessing medical advice and using treatment for infertility were identified for women aged less than 34 years. Among a community sample of infertile women aged 28-33 years (ALSWH participants), self-reported depression was found to be a barrier to accessing medical advice. The characteristics of these infertile women in the community who had (n=121) or had not (n=110) used treatment were compared to infertile women aged 27-33 years (n=59) attending four fertility clinics. Compared to infertile women in the community, living in major cities and having private health insurance were associated with early use of treatment for infertility at specialist clinics by women aged <34 years. In contrast to most clinical studies of IVF, the final study reported in the thesis took into account repeated IVF cycles and the impact of women’s individual histories on IVF outcomes. Among 121 infertile women (aged 27-46 years) who had 286 IVF cycles, older age and prolonged use of the oral contraceptive pill were associated with fewer eggs collected. Further, women in particular occupations had lower proportions of eggs fertilised normally than women in other occupational groups. These studies form the first large-scale epidemiological examination of infertility in Australia. The finding that two-thirds of women with infertility had not used treatment indicates that there is an unmet need for specialist treatment in women aged less than 34 years. However, barriers to accessing treatment prevent women using ART at a younger age when there is a higher chance of pregnancy.

Studies of assisted reproduction in the spotted grass frog Limnodynastes tasmaniensis: ovulation, early development and microinjection (ICSI)

Assisted Reproductive Technologies (ART) offer a wide range of techniques that have the potential to augment efforts to conserve and manage endangered amphibians and improve wild and captive population numbers. Gametes and tissues of species nearing endangered or extinct status can be cryopreserved and stored in gene banks, to provide material that can be utilised in the future as ART methods are refined. The Spotted Grass Frog, Limnodynastes tasmaniensis, is an abundant amphibian species in South-Eastern Australia of the family Myobatrachidae, that is suitable for the development of ART systems that can be applied to the threatened and endangered myobatrachid and other amphibian species native to Australia. The aim of this study was to advance the understanding of ovulation, fertilisation and embryo nic development of Lim. tasmaniensis and in vitro manipulations of reproduction and development for use in the development of advanced ART procedures such as intracytoplasmic spermatozoon injection (ICSI), androgenesis and nuclear transfer. Ovulation in amphibians can be induced by protocols utilising natural or synthetic hormones. All protocols tested on Lim. tasmaniensis in this study required two injections and the most effective protocols continued to require a first injection of pituitary extracts to induce ovulation. The second injection was, however, successfully replaced by synthetic chorionic gonadotrophin at a threshold dosage of 100 iu and halved the number of cane toads required to source the pituitaries. A combination of LHRH and Pimozide offered a less effective protocol, that did not require the use of pituitary extracts, and avoided the risk of pathogen transfer associated with unsterilised pituitary extracts. Unfertilised eggs of Lim. tasmaniensis were exposed to media of various osmolalities to determine media effects on eggs and their surrounding jelly layers that might impact on egg viability and fertilisability. Osmolality had no effect upon the egg diameter, however, rapid swelling of the jelly layers occurred within 15 minutes of exposure to various media treatments and plateaued from 30-90 minutes without further expansion. Swelling of the jelly layers was increased in hypotonic media (2.5% SAR, H2O) and minimised in the isotonic media (100% SAR). The optimal conditions for the culture of Lim. tasmaniensis eggs were identified as a holding media of 100% SAR, followed by a medium change to 2.5% SAR at insemination. This sequence of media minimised the rate of swelling of the jelly layers prior to contact with the spermatozoa, and maximised the activation of spermatozoa and eggs throughout fertilisation and embryonic development. Embryos of Lim. tasmaniensis were cultured at four temperatures (13 C, 17 C, 23 C and 29 C), to determine the effect of temperature on cleavage and embryonic development rates. Embryonic development progressed through a sequence of stages that were not altered by changes in temperature. However cleavage rates were affected by changes in temperature as compared with normal embryonic growth at 23 C. Embryonic development was suspended at the lowest temperature (13 C) while embryonic viability was maintained. A moderate decrease in temperature (17 C) slowed cleavage, while the highest temperature (29 C) increased the cleavage rate, but decreased the embryo survival. Rates of embryonic development can be manipulated by changes in temperature and this method can be used to source blastomeres of a specific size/stage at a predetermined age or halt cleavage at specific stages for embryos or embryo derived cells to be included in ART procedures. This study produced the first report of the application of Intracytoplasmic Spermatozoon Injection (ICSI) in an Australian amphibian. Eggs that were activated by microinjection with a single spermatozoon (n=50) formed more deep, but abnormal, cleavage furrows post-injection (18/50, 36%), than surface changes (12/50, 24%). This result is in contrast to eggs injected without a spermatozoon (n=42), where the majority of eggs displayed limited surface changes (36/42, 86%), and few deep, abnormal furrows (3/42, 7%). Three advanced embryos (3/50, 6%) were produced by ICSI that developed to various stages within the culture system. Technical difficulties were encountered that prevented the generation of any metamorphs from ICSI tadpoles. Nevertheless, when these blocks to ICSI are overcome, the ICSI procedure will be both directly useful as an ART procedure in its own right, and the associated refinement of micromanipulation procedures will assist in the development of other ART procedures in Lim. tasmaniensis. A greater understanding of basic reproductive and developmental biology in Lim. tasmaniensis would greatly facilitate refinement of fertilisation by ICSI. Assisted Reproductive Technologies, in conjunction with gene banks may in the future regenerate extinct amphibian species, and assist in the recovery of declining amphibian populations nationally and worldwide.