Effects of dietary supplementation of different oils and conjugated linoleic acid on the reproductive and metabolic aspects of male mice.

The present study was carried out to examine first, if diets enriched with 320 g of the base diet with common dietary oils including fish oil, olive oil, hydrogenated sunflower seed (H-SFS) oil, flaxseed oil and sunflower seed oil (SFS) could induce weight gain and alter reproductive and metabolic characteristics of male mice. Second, whether the addition of conjugated linoleic acid (CLA, 10% of the diet) could ameliorate any negative effects. In this cross-sectional study, 90 four-week-old male NMRI mice were used in two consecutive experiments. A high level of dietary oils negatively affected some reproductive and metabolic characteristics of male mice (p < 0.05), specifically, sunflower seed oil enrichment resulted in higher HDL levels and apoptosis of germinal epithelial cells. An olive oil-enriched diet caused an increase in plasma triglyceride concentrations and germinal cell apoptosis, as well as a decrease in sperm concentration and perturbed spermatogenesis. When CLA was fed in conjunction with dietary oils it successfully mitigated some of the negative reproductive and metabolic characteristics. We conclude that male reproductive processes are affected by high dietary oils, even before signs of obesity are evident. Inclusion of dietary CLA may provide some benefit to offset negative effects, although further studies are required.

Podocalyxin molecular characteristics and endometrial expression: high conservation between humans and macaques but divergence in mice†.

Podocalyxin (PODXL) is a newly identified key negative regulator of human endometrial receptivity, specifically down-regulated in the luminal epithelium at receptivity to permit embryo implantation. Here, we bioinformatically compared the molecular characteristics of PODXL among the human, rhesus macaque, and mouse, determined by immunohistochemistry and in situ hybridization (mouse tissues) whether endometrial PODXL expression is conserved across the three species and examined if PODXL inhibits mouse embryo attachment in vitro. The PODXL gene, mRNA, and protein sequences showed greater similarities between humans and macaques than with mice. In all species, PODXL was expressed in endometrial luminal/glandular epithelia and endothelia. In macaques (n = 9), luminal PODXL was significantly down-regulated when receptivity is developed, consistent with the pattern found in women. At receptivity, PODXL was also reduced in shallow glands, whereas endothelial expression was unchanged across the menstrual cycle. In mice, endometrial PODXL did not vary considerably across the estrous cycle (n = 16); however, around embryo attachment on d4.5 of pregnancy (n = 4), luminal PODXL was greatly reduced especially near the site of embryo attachment. Mouse embryos failed to attach or thrive when co-cultured on a monolayer of Ishikawa cells overexpressing PODXL. Thus, endometrial luminal PODXL expression is down-regulated for embryo implantation in all species examined, and PODXL inhibits mouse embryo implantation. Rhesus macaques share greater conservations with humans than mice in PODXL molecular characteristics and regulation, thus represent a better animal model for functional studies of endometrial PODXL for treatment of human fertility.

The impact of COVID-19 mitigation measures on fertility patients and clinics around the world.

RESEARCH QUESTION: What is the impact of the response to COVID-19 on the management of fertility treatments and clinical practice around the world? DESIGN: Fertility clinic associates around the world were approached. They completed an online survey containing 33 questions focused on the country's response to the COVID-19 pandemic. Known fertility clinic associates that were contacted comprised scientific directors, medical directors and laboratory managers. RESULTS: There were 43 individual country responses from Asia (13), Africa (3), Europe (17), North America (3), Oceania (2) and South America (5). In nine countries, clinics followed their government body recommendations, in 22 countries there was a combination of recommendations, in 3 countries changes were made by clinic initiative, and 9 countries did not specify. In 34 countries IVF/intracytoplasmic sperm injection (ICSI) and frozen embryo transfer (FET) treatments had an average delay of 56 days (IVF/ICSI) (minimum 0, maximum 160) and 57 days (FET) (minimum 0, maximum 166 days). During the shutdown, the number of freeze-all cycles increased in 22 countries. Only 23 countries reported patients having to undergo a SARS-CoV-2 test, and 20 countries did not report any COVID-19 testing in their clinic. Additional support counselling was offered in 28 countries, partner restrictions at clinics were reported in 41 countries and time between patients' appointments was increased in 39 countries. CONCLUSIONS: The implications of COVID-19 mitigation measures proved the need for government societies to introduce a set protocol that includes requirements such as increased patient counselling and additional guidelines for prioritizing couples who need care most urgently.

Financing future fertility: Women's views on funding egg freezing.

Like other assisted reproductive technology (ART) procedures, the cost of egg freezing (EF) is significant, presenting a potential barrier to access. Given recent technological advancements and rising demand for EF, it is timely to reassess how EF is funded. An online cross-sectional survey was conducted in Victoria, Australia and was completed by 656 female individuals. Participants were asked their views on funding for both medical and non-medical EF. The median age of participants was 28 years (interquartile range 23-37 years) and most participants were employed (44% full-time, 28% part-time, 33% students). There was very high support for public funding for medical EF (n = 574, 87%), with 302 (46%) participants indicating support for the complete funding of medical EF through the public system. Views about funding for non-medical EF were more divided; 43 (6%) participants supported full public funding, 235 (36%) supported partial public funding, 150 (23%) supported coverage through private health insurance, and 204 (31%) indicated that non-medical EF should be self-funded. If faced with the decision of what to do with surplus eggs, a high proportion of participants indicated that they would consider donation (71% to research, 59% to a known recipient, 52% to a donor programme), indicating that eggs surplus to requirements could be a potential source of donor eggs. This study provides insights that could inform policy review, and suggests revisiting whether the medical/non-medical distinction is a fair criterion to allocate funding to ART.

Employer-Sponsored Egg Freezing: Carrot or Stick?

BackgroundSince 2014, many companies have followed the lead of Apple and Facebook and now offer financial support to female employees to access egg freezing. Australian companies may soon make similar offers. Employer-sponsored egg freezing (ESEF) has raised concerns and there is academic debate about whether ESEF promotes reproductive autonomy or reinforces the 'career vs. family' dichotomy. Despite the growing availability of ESEF and significant academic debate, little is known about how ESEF is perceived by the public. The aim of this study was to explore women's attitudes toward ESEF.MethodsWomen aged 18-60 years who resided in Victoria, Australia were invited to complete an online, cross-sectional survey investigating views toward egg freezing. Associations between participant demographics and their views about ESEF were assessed using multinominal logistic regression, adjusted for age and free text comments were analyzed using thematic analysis.ResultsThe survey was completed by 656 women, median age 28 years (range: 18-60 years). Opinions on the appropriateness of employers offering ESEF were divided (Appropriate: 278, 42%; Inappropriate: 177, 27%; Unsure: 201, 31%). There was significantly less support for ESEF among older participants and those employed part-time (p < 0.05). While some participants saw the potential for ESEF to increase women's reproductive and career options, others were concerned that ESEF could pressure women to delay childbearing and exacerbate existing inequities in access to ARTs.ConclusionsOur analysis revealed that while some women identified risks with ESEF, for many women ESEF is not viewed as theoretically wrong, but rather it may be acceptable under certain conditions; such as with protections around reproductive freedoms and assurances that ESEF is offered alongside other benefits that promote career building and family. We suggest that there may be a role for the State in ensuring that these conditions are met.

A major increase in oocyte cryopreservation cycles in the USA, Australia and New Zealand since 2010 is highlighted by younger women but a need for standardized data collection.

STUDY QUESTION: What are the cohort trends of women undergoing oocyte cryopreservation (OC)? SUMMARY ANSWER: There has been a dramatic increase in OC cycles undertaken each year since 2010, and the demographics of women accessing OC has shifted to a younger age group, but so far very few women have returned to use their cryopreserved oocytes in treatments. WHAT IS KNOWN ALREADY: Although OC, as a method of fertility preservation, is offered around the world, global data are lacking on who is accessing OC, who is returning to thaw oocytes and whether these trends are changing. STUDY DESIGN, SIZE, DURATION: A trinational retrospective cohort study was performed of 31 191 OC cycles and 972 oocyte thaw (OT) cycles undertaken in the USA (2010-2016) and 3673 OC and 517 OT cycles undertaken in Australia/New Zealand (Aus/NZ; 2010-2015). PARTICIPANTS/MATERIALS, SETTING, METHODS: Data were obtained from the USA Society for Assisted Reproductive Technology (SART) national registry and the Australian and New Zealand Assisted Reproduction Database (ANZARD). De-identified data were requested on all autologous oocyte freeze-all cycles and all cycles where autologous oocytes were thawed to be used in a treatment cycle for the time periods of interest. MAIN RESULTS AND THE ROLE OF CHANCE: In both the USA and Aus/NZ, there has been a dramatic rise in the number of OC cycles performed each year (+880% in the USA from 2010 to 2016 and +311% in Aus/NZ from 2010 to 2015). Across both regions, most women undergoing OC were aged in their late 30s, but the average age decreased over time (USA: 36.7 years vs 34.7 years in 2010 and 2016, respectively). The number of women returning for thaw cycles was low (USA: 413 in 2016, Aus/NZ: 141 in 2015) and most thaw cycles (47%) across both regions involved oocytes that were frozen for <6 months. In the USA, a higher proportion of cycles resulted in a live birth when only thawed oocytes were used, compared to cycles that combined thawed oocytes with fresh oocytes (25% vs 11%, respectively; P < 0.001). Age at retrieval influenced live birth rate in the USA; 38% of thaw cycles started in women who stored oocytes when aged ≤35 years resulted in a live birth, whereas only 16% resulted in a live birth for women who stored oocytes when aged ≥36 years. Similar data were unobtainable from Aus/NZ. LIMITATIONS, REASONS FOR CAUTION: There were limitations associated with both the SART and ANZARD data outputs received. The format in which the ANZARD data were provided, and the inconsistencies seen amongst cycle reporting in the SART dataset, restricted data interpretation. For example, both datasets did not provide a clear indication as to why women were undergoing OC and it was not possible to accurately calculate duration of storage for thaw cycles in the USA. We also did not obtain details on embryo quality from either database and acknowledge that embryo quality and subsequent outcome (embryo freezing or discard) would be of interest, especially when considering the efficacy of OC. WIDER IMPLICATIONS OF THE FINDINGS: The data show that there is widespread demand for OC, and it is increasingly undertaken by younger women; however, the limitations encountered in the dataset support the need for a shift to a more uniform approach to data collection and presentation by large databases, worldwide. STUDY FUNDING/COMPETING INTEREST(S): This study received funding from the Fertility Society of Australia to support the ANZARD data extraction. M.J. is supported by an Australian Government Research Training Program Scholarship stipend. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Ageing and ovarian stimulation modulate the relative levels of transcript abundance of oocyte DNA repair genes during the germinal vesicle-metaphase II transition in mice.

PURPOSE: 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. METHODS: 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). RESULTS: 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). CONCLUSIONS: 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.

A human-based assisted reproduction protocol for the menstruating spiny mouse, Acomys cahirinus.

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.

Successful sperm cryopreservation in Egyptian spiny mice Acomys cahirinus.

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.

Cracked open: exploring attitudes on access to egg freezing.

Egg freezing (EF) technology has improved significantly over the last decade, giving women more choice over their reproductive futures. Despite this advance, EF brings forth contentious ethical and regulatory issues. Policies controlling access to EF vary around the world and there is a lack of consensus about who should have access and what criteria are relevant in making these decisions. This study aimed to identify views of women about access to EF for both "medical" and "non-medical" risks to infertility. An online survey was administered to women aged between 18 and 60 years in Victoria, Australia between April and May 2018. A total of 1,066 individuals initiated the survey. The median age of the participants was 28 years and 81% were <40 years old. Almost all participants (98%) supported access to medical EF in situations where treatments (e.g. chemotherapy) or illnesses threaten fertility. Support for access to EF for non-medical indications was lower; 75% supported EF for "lack of suitable partner", followed by "financial insecurity to raise a child" (72%) and "career/educational advancement" (65%). Older respondents (aged ≥40 years) were less likely than their younger counterparts to support all indications for non-medical EF. Our findings indicate broad support for EF. However, the variation in support between indications for non-medical EF suggests that individuals do not think about access to EF simply in terms of medical necessity. To reflect public views, future policy may need to consider access to EF beyond the medical/non-medical distinction.

Tolerance of lamb and mouse oocytes to cryoprotectants during vitrification.

SummaryMouse and lamb oocytes were vitrified with, or exposed to, different cryoprotectants and evaluated for their effects on their survival and developmental competence after in vitro fertilization (IVF) and activation treatments. Control oocytes remained untreated, whilst the remainder were exposed to three different combinations of vitrification solutions [dimethyl sulfoxide (DMSO) + ethylene glycol (EG), EG only, or propanediol (PROH) + EG] and either vitrified or left unfrozen (exposed groups). Oocytes in the control and vitrified groups underwent IVF and developmental competence was assessed to the blastocyst stage. In lambs, survival rate in vitrified oocytes was significantly lower than for oocytes in the exposed groups (P <0.05). Blastocyst development was low in vitrified oocytes compared with controls (<6% vs 38.9%, P <0.01). Parthenogenetic activation was more prevalent in vitrified lamb oocytes compared with controls (P <0.05). No evidence of zona pellucida hardening or cortical granule exocytosis could account for reduced fertilization rates in vitrified lamb oocytes. Mouse oocytes demonstrated a completely different response to lamb oocytes, with survival and parthenogenetic activation rates unaffected by the vitrification process. Treatment of mouse oocytes with DMSO + EG yielded significantly higher survival and cleavage rates than treatment with PROH + EG (87.8% and 51.7% vs 32.7% and 16.7% respectively, P <0.01), however cleavage rate for vitrified oocytes remained lower than for the controls (51.7% vs 91.7%, P <0.01) as did mean blastocyst cell number (33 ± 3.1 vs 42 ± 1.5, P <0.05). From this study, it is clear that lamb and mouse show different tolerances to cryoprotectants commonly used in vitrification procedures, and careful selection and testing of species-compatible cryoprotectants is required when vitrifying oocytes to optimize survival and embryo development.

Assessment of potential biomarkers of pre-receptive and receptive endometrium in uterine fluid and a functional evaluation of the potential role of CSF3 in fertility.

The endometrium lines a women's uterus becoming receptive, and allowing embryo implantation to occur, for just a few days during the post-ovulatory mid-secretory phase of each menstrual cycle. We investigated whether concentrations of proposed receptivity biomarkers (VEGF, IL8, FGF2, CSF3 sFlt-1, sGP130 and PlGF) secreted by the endometrium into the uterine cavity and forming the microenvironment for embryo implantation is altered among a population of age-matched women with unexplained (idiopathic) infertility compared to fertile women during the receptive mid-secretory phase (n = 16 fertile, 18 infertile) and the prior pre-receptive early secretory phase (n = 19 fertile, 18 infertile) of their cycle. In the mid-secretory cohort significantly elevated concentrations of five biomarkers; PlGF (p = 0.001), IL8 (p = 0.004), sGP130 (p = 0.009), sFlt-1 (p = 0.021), and CSF3 (p = 0.029) was present in uterine fluid of infertile women during the mid-secretory phase, but only CSF3 was significantly elevated in the pre-receptive early secretory phase (p = 0.006). In vitro studies of glycosylated and non-glycosylated forms of CSF3 at representative fertile (20 ng/mL) and infertile (70 ng/mL) effects on endometrium and embryo behaviour were performed. Non-glycosylated CSF3 at fertile concentrations significantly (p < 0.001) elevated endometrial epithelial cell proliferation however chronic treatment or elevated (infertile) concentrations of CSF3 in glycosylated form abrogated the positive effects. Both forms of CSF3 increased trophoblast cell invasion (p < 0.001) regardless of concentration. Mouse embryo outgrowth was significantly (p < 0.01) increased at fertile but not at infertile concentrations. The study confirmed potential utility of five biomarkers of endometrial receptivity for future application in the mid-secretory phase while highlighting CSF3 is elevated in the earlier pre-receptive phase. Our data provides evidence that CSF3 acts on both human endometrium and embryo in a manner that is concentration and glycosylation dependent.

Comparison of pregnancy in cattle when non-vitrified and vitrified in vitro-derived embryos are transferred into recipients.

The present study was conducted in cattle to test the null hypothesis that the pregnancy rate of recipient females is similar when in vitro-derived embryos are transferred either fresh (non-vitrified) or after being subjected to vitrification. Cumulus-oocyte complexes, collected twice (6 weeks apart) from 10 donor cows were matured in vitro and inseminated with frozen-thawed sperm from a single proven bull per donor collection. Cleaved embryos were cultured in vitro until day 7 and any resulting blastocysts were graded for stage [early (unexpanded), advanced (expanded, hatching, hatched)] and/or quality and either discarded (poor quality), or, if deemed suitable, transferred fresh or vitrified for later warming and transfer. All blastocysts were transferred singly to oestrus-synchronized cows and pregnancy monitored by transrectal palpation on days 35, 60 and 90. From 20 collections, 818 cumulus-oocyte complexes were aspirated; however, after grading, only 462 (56.5%) were ranked as suitable quality for maturation and insemination. From those 462 complexes inseminated, 363 (78.6%) cleaved during the process and 243 (52.6%) developed to the blastocyst stage with 194 (42.0%) deemed utilizable, of which 85 were vitrified and 109 were transferred fresh. There was a median of 13 (range 0-24) utilizable blastocysts per cow. Of the 109 non-vitrified blastocysts transferred, there were 45 (41.3%) and 41 (37.6%) recipients that were detected to be pregnant on day 35 and day 90, respectively, subsequent to transfer. Thus, an 8.9% abortion rate was observed (4/45). Of the 85 transferred vitrified-warmed blastocysts, 34 were detected to be pregnant (40.0%) on day 35 following transfer, and all pregnancies were maintained at day 90 (0% abortion rate), which was similar to non-vitrified transfers (P > 0.05, Chi-square test). There was no significant difference in pregnancy rate on day 90 in advanced compared to early blastocysts for either the non-vitrified transfers (9/23, 39.1% vs 33/86, 38.3%) or the vitrified transfers (30/72, 41.6% vs 4/13, 30.8%) (P > 0.05 in each case). In summary, these data show that vitrification of in vitro-derived early and advanced blastocysts is a suitable method of cryopreservation of bovine embryos, and, furthermore, that subsequent transfer of all vitrified/warmed blastocysts into recipient females results in pregnancy rates no different to those attained by non-vitrified transfers into recipient females.

A comparative analysis of the efficacy of three cryopreservation protocols on the survival of in vitro-derived cattle embryos at pronuclear and blastocyst stages.

The effectiveness of three cryopreservation protocols (slow freezing, short equilibration vitrification and long equilibration vitrification) on in vitro-derived cattle embryos at expanded blastocyst and pronuclear stages was compared. 199 expanded blastocysts of good quality were assigned randomly into four treatment groups [control, non-cryopreserved (fresh, unfrozen); and the three cryopreservation methods]. The re-expansion of the cryopreserved blastocysts after 24 h in vitro culture was similar to that of the fresh control group. However, the hatching rate of expanded blastocysts after 48 h culture was significantly less for the slow freezing group (31/47; 66.0%) than for both the short equilibration vitrification (46/51; 90.2%) and long equilibration vitrification groups (42/50; 84.0%). Denuded presumptive zygotes at the pronuclear stage (14-18 h post-insemination) were assigned randomly to the same four treatment groups and, following thawing, embryos were assessed for their capacity to cleave and to develop into a blastocyst. Overall, cleavage rates of cryopreserved zygotes were significantly less than those of the fresh control. The blastocyst formation rate of slow-frozen zygotes (4/81; 4.9%) was significantly less than that of zygotes subjected either to short equilibration vitrification (18/82; 22.0%) or long equilibration vitrification (16/74; 21.6%). All cryopreservation groups showed rates of blastocyst formation that were significantly less than that of the fresh control (51/92; 55.4%). Collectively, our findings indicate that vitrification is the preferred technology to cryopreserve in vitro-derived cattle embryos at expanded blastocyst and pronuclear stages. Moreover, short equilibration vitrification technology can improve outcomes and be more efficient by taking less time to perform.

Minimal volume vitrification of epididymal spermatozoa results in successful in vitro fertilization and embryo development in mice.

This study compared three cryopreservation protocols on sperm functions, IVF outcomes, and embryo development. Epididymal spermatozoa cryopreserved using slow-cooling (18% w/v raffinose, RS-C) were compared with spermatozoa vitrified using 0.25 M sucrose (SV) or 18% w/v raffinose (RV). The motility, vitality, and DNA damage (TUNEL assay) of fresh control (FC) spermatozoa were compared with post-thawed or warmed RS-C, RV, and SV samples. Mouse oocytes (n = 267) were randomly assigned into three groups for insemination: RV (n = 102), RS-C (n = 86), and FC (n = 79). The number and the proportion of two-cell embryos and blastocysts from each treatment were assessed. Sperm motility (P < 0.01) and vitality (P < 0.05) were significantly reduced after vitrification compared with slow-cooled spermatozoa. However, DNA fragmentation was significantly reduced in spermatozoa vitrified using sucrose (15 ± 1.8% [SV] vs 26 ± 2.8% [RV] and 27 ± 1.2% [RS-C]; P < 0.01). Although the number of two-cell embryos produced by RS-C, RV, and FC spermatozoa was not significantly different, the number of blastocysts produced from two-cell embryos using RV spermatozoa was significantly higher than FC spermatozoa (P = 0.0053). This simple, small volume vitrification protocol and standard insemination method allows successful embryo production from small numbers of epididymal spermatozoa and may be applied clinically to circumvent the need for ICSI, which has the disadvantage of bypassing sperm selection.

Characterisation of the cellular and molecular responses of ovine oocytes and their supporting somatic cells to pre-ovulatory levels of LH and FSH during in vitro maturation.

The response of Graafian follicles to pre-ovulatory surge levels of FSH and LH in vivo triggers the terminal differentiation of granulosa cells and oocyte maturation. In polyovular species, the LH-driven signalling uses the epidermal growth factor (EGF)-like ligands AREG, EREG and BTC to promote oocyte maturation and cumulus expansion. This experimental series used a physiologically relevant ovine in vitro maturation (IVM) system to evaluate the impact of exposure to pre-ovulatory levels (100  ng/ml) of LH and FSH on ovine cumulus cell expression of EGF-like ligands in vitro. The serum-free sheep IVM system supported high levels (91.4%) of gonadotrophin-induced maturation of cumulus-enclosed oocytes and embryo development to the blastocyst stage (34.5%). Results were equivalent to a serum-based IVM system (85.1% IVM, 25.8% blastocyst rate; P>0.05) but were significantly different (P<0.05) to serum-free medium without gonadotrophins (69.5% IVM; 8.0% blastocyst rate). Ovine BTC was cloned and sequenced. Gonadotrophin-induced AREG, EREG, BTC and EGFR expressions were quantified in cumulus and mural granulosa cells during IVM. A rapid induction of AREG expression was apparent in both cell types within 30  min of gonadotrophin exposure in vitro. LHCGR (LHR) was detected in mural cells and FSHR in both cumulus and mural granulosa cells. The data confirm the involvement of AREG and EGFR during gonadotrophin-induced cumulus expansion, oocyte maturation and the acquisition of developmental competence by sheep oocytes matured in vitro.

Successful in vitro culture of pre-antral follicles derived from vitrified murine ovarian tissue: oocyte maturation, fertilization, and live births.

Cryopreservation of ovarian tissue is an important option for preserving the fertility of cancer patients undergoing chemotherapy and radiotherapy. In this study, we examined the viability and function of oocytes derived in vitro from pre-antral follicles as an alternative method for restoring fertility. Pre-antral follicles (specified as secondary follicle with a diameter around 100-130 µm) were mechanically isolated from vitrified-warmed and fresh adult mouse ovarian tissues and cultured for 12 days followed by an ovulation induction protocol at the end of this period to initiate oocyte maturation. Oocytes were then released from these follicles, fertilized in vitro, and cultured to the blastocyst stage and vitrified. After storage in liquid nitrogen for 2 weeks, groups of vitrified blastocysts were warmed and transferred into pseudo-pregnant recipient females. Although most of the isolated mouse pre-antral follicles from fresh (79.4%) and vitrified (75.0%) ovarian tissues survived the 12-day in vitro culture period, significantly fewer mature oocytes developed from vitrified-warmed pre-antral follicles than from the fresh controls (62.2 vs 86.4%, P<0.05). No difference was observed in embryo cleavage rates between these two groups, but the proportion of embryos that developed into blastocysts in the vitrification group was only half that of the controls (24.2 vs 47.2%, P<0.05). Nevertheless, live births of healthy normal pups were achieved after transfer of vitrified blastocysts derived from both experimental groups. This study shows that successful production of healthy offspring using an in vitro follicle culture system is feasible, and suggests that this procedure could be used in cancer patients who wish to preserve their fertility using ovarian tissue cryopreservation.

Live offspring from vitrified blastocysts derived from fresh and cryopreserved ovarian tissue grafts of adult mice.

Ovarian tissue cryopreservation and transplantation can be used to preserve fertility for cancer patients. In this study, we assessed the viability and function of ovarian tissue from adult mice that was cryopreserved by solid surface vitrification or traditional slow-cooling using various in vitro and in vivo techniques, including allotransplantation, in vitro oocyte maturation, embryo culture in vitro, blastocyst cryopreservation, embryo transfer, and development. The importance of cumulus cells for oocyte maturation, fertilization, and embryo development was investigated. Graft recovery, follicle survival, and oocyte retrieval was similar in control, vitrified, and slow-cooled groups. High rates of oocyte maturation, cleavage, and blastocyst formation were achieved, with no significant differences between the control, vitrified or slow-cooled ovarian tissue grafts. The presence of cumulus cells was important for oocyte maturation, fertilization, and subsequent development. Cumulus-oocyte complexes with no surrounding cumulus cells (N-COCs) or with an incomplete layer (P-COCs) had significantly lower rates of oocyte maturation and blastocyst formation than cumulus-oocyte complexes with at least one complete layer of cumulus cells (F-COCs; maturation rate: 63, 78 vs 94%; blastocyst rate: 29, 49 vs 80%). Live births were achieved using vitrified blastocysts derived from oocytes taken from vitrified and slow-cooled ovarian tissue heterotypic allografts. Successful production of healthy offspring from these vitrified blastocysts suggests that this technique should be considered as a useful stage to pause in the assisted reproduction pathway. This provides an alternative protocol for restoring fertility and offering cancer patients a better indication of their chances of pregnancy and live birth.

Effects of lamb age, hormone stimulation and response to hormone stimulation on the yield and in vitro developmental competence of prepubertal lamb oocytes.

Experiments were conducted to determine the effects of lamb age, hormone stimulation (Experiment 1) and response to stimulation (Experiment 2) on the in vitro production of embryos from prepubertal lambs aged 3-4 and 6-7 weeks of age. For 3-4-week-old lambs, hormone stimulation increased the number of follicles (29.9 +/- 15.3 v. 70.6 +/- 8.2), oocytes per ovary (18.3 +/- 6.3 v. 39.3 +/- 5.8) and oocyte development to the blastocyst stage (0/192 (0.0%) v. 115/661 (17.4%); P < 0.05). Lamb age (3-4 v. 6-7 weeks old) increased oocyte development to the blastocyst stage (115/661 (17.4%) v. 120/562 (21.4%) respectively). In Experiment 2, hormone-stimulated lambs (3-4 and 6-7 weeks old) were divided into low, medium or high responders based on the number of ovarian follicles (<20, 20-50 and >100 follicles per ovary respectively). The response to hormone stimulation did not affect oocyte recovery rate, but the number of oocytes suitable for culture was increased for high-responding 3-4-week-old lambs only (P < 0.05). Oocyte development to the blastocyst stage was not affected by response to stimulation for 3-4-week-old lambs (15.2-25.6%; P > 0.05), but was reduced for high (6.7%) compared with low (19.5%) and medium (30.9%) responding 6-7-week-old lambs (P < 0.05). These results demonstrate that the production of embryos from prepubertal lambs is increased by hormone stimulation and lamb age and the response to stimulation does not affect embryo production from 3-4-week-old lambs, although by 6-7 weeks of age a high response to stimulation reduces blastocyst formation.

In vitro and in vivo developmental capabilities and kinetics of in vitro development of in vitro matured oocytes from adult, unstimulated and hormone-stimulated prepubertal ewes.

The in vitro and in vivo developmental capabilities and kinetics of in vitro development of embryos derived from adult ewes and from unstimulated (16- to 24-week-old) and hormone-stimulated prepubertal (3- to 5-week-old) ewes were assessed. Cleavage was lower for hormone-stimulated (617/1025; 60.2%) than unstimulated prepubertal (117/169; 69.2%) and adult ewe oocytes (184/267; 68.9%; P < 0.05). Blastocyst formation by Day 7 (from zygotes) was similar for unstimulated (45/117; 38.5%), hormone-stimulated prepubertal (229/617; 37.1%) and adult ewes (101/184; 54.9%). Blastocysts derived from hormone-stimulated prepubertal ewes developed mainly on day 7, compared with Day 6 for adult and unstimulated prepubertal ewes. Pregnancy rates (day 60) and embryonic loss (between Days 20 and 60) did not differ after transfer to adult recipient ewes of adult, unstimulated and hormone-stimulated prepubertal-derived fresh or frozen-thawed embryos. The number of lambs born as a proportion of embryos transferred did not differ for fresh and frozen embryos derived from adult ewes (3/16; 18.8% and 1/12; 8.3%, respectively) and unstimulated prepubertal lambs (2/6; 33.3%, and 1/10; 10.0%, respectively), but was higher for fresh than frozen embryos from hormone-stimulated prepubertal ewes (7/16; 43.8%, and 2/14; 14.3%, respectively; P < 0.05). There were high rates of in vitro and in vivo development of oocytes from 3- to 5-week-old lambs, but in vitro development was lower than with oocytes from adult ewes. However, the speed of embryonic development in vitro and the in vivo development of fresh and frozen embryos were similar to those derived from adult and unstimulated prepubertal ewes. The present results were an improvement in the efficiency of producing embryos and offspring from hormone-stimulated 3- to 5-week-old lambs.