Dropout rate and cumulative birth outcomes in couples undergoing in vitro fertilization within a funded and actively managed system of care in New Zealand.

OBJECTIVE: To determine the dropout rate between the first and second in vitro fertilization (IVF) cycles in a controlled population derived from a funded and actively managed system of care in New Zealand, including the reason for dropout and associated cumulative live birth rate. DESIGN: Retrospective cohort. SETTING: Multicenter IVF practice. PATIENT(S): Couples qualifying for publicly funded IVF treatment under New Zealand's Clinical Priority Assessment Criteria. Couples (n = 974) started treatment between July 2011 and June 2013, used their own gametes, and were eligible for up to 2 IVF packages of funded care (including the transfer of surplus embryos). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): IVF dropout rate, reason for dropout, and cumulative live birth rate. RESULT(S): A low IVF dropout rate between the first and second IVF cycle was reported within this controlled IVF population, with 10% of couples discontinuing treatment for reasons related to stress. The cumulative live birth rate in this "low dropout" population was 59% at the end of treatment, ranging from 72% (≤30 years) to 42% (38-39 years) according to female age. Most patients who discontinued for stress had a good prognosis, and a third of patients still had embryos in cryostorage. Only 30% of those who discontinued used the funded counseling services. CONCLUSION(S): A low dropout rate (10%) can be achieved within an actively managed IVF population. This was lower than previously reported, suggesting that prognosis, cost, and treatment management are the significant causes of dropout within the general IVF population. Couples with many embryos also require psychological support because of treatment fatigue or repeated transfers.

Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified.

OBJECTIVE: To characterize nuclear and mitochondrial DNA (mtDNA) in spent culture media from normally developing blastocysts to determine whether it could be used for noninvasive genetic assessment. DESIGN: Prospective embryo cohort study. SETTING: Academic center and private in vitro fertilization (IVF) clinic. PATIENT(S): Seventy patients undergoing intracytoplasmic sperm injection (ICSI) and 227 blastocysts. INTERVENTION(S): Culture media assessment, artificial blastocoele fluid collapse and DNA analysis using digital polymerase chain reaction (dPCR), long-range PCR, quantitative PCR (qPCR), and DNA fingerprinting. MAIN OUTCOME MEASURE(S): Presence of nuclear and mtDNA in three different commercial culture media from Vitrolife and Irvine Scientific, spent embryo media assessment at the cleavage and blastocyst stages of development, and analysis of the internal media controls for each patient that had been exposed to identical conditions as embryo media but did not come into contact with embryos. RESULT(S): Higher levels of nuclear and mtDNA were observed in the culture media that had been exposed to embryos compared with the internal media controls. Nuclear DNA (∼4 copies) and mtDNA (∼600 copies) could be detected in spent media, and the levels increased at the blastocyst stage. No increase in DNA was detected after artificial blastocoele fluid collapse. Mixed sex chromosome DNA was detected. This originated from contamination in the culture media and from maternal (cumulus) cells. Due to the limited amount of template, the presence of embryonic nuclear DNA could not be confirmed by DNA fingerprinting analysis. CONCLUSION(S): Currently DNA from culture media cannot be used for genetic assessment because embryo-associated structures release DNA into the culture medium and the DNA is of mixed origin.

Oocyte mitochondrial deletions and heteroplasmy in a bovine model of ageing and ovarian stimulation.

STUDY HYPOTHESIS: Maternal ageing and ovarian stimulation result in the accumulation of mitochondrial DNA (mtDNA) deletions and heteroplasmy in individual oocytes from a novel bovine model for human assisted reproductive technology (ART). STUDY FINDING: The levels of mtDNA deletions detected in oocytes increased with ovarian ageing. Low levels of mtDNA heteroplasmy were apparent across oocytes and no relationship was identified with respect to ovarian ageing or ovarian stimulation. WHAT IS KNOWN ALREADY: Oocyte quality decreases with ovarian ageing and it is postulated that the mtDNA may have a role in this decline. The impact of ovarian stimulation on oocyte quality is poorly understood. Human studies investigating these effects are often limited by the use of low quality oocytes and embryos, variation in age and ovarian stimulation regimens within the patients studied, as well as genetic and environmental variability. Further, no study has investigated mtDNA heteroplasmy in individual oocytes using next-generation sequencing (NGS), and little is known about whether the oocyte accumulates heteroplasmic mtDNA mutations following ageing or ovarian stimulation. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: A novel bovine model for the effect of stimulation and age in human ART was undertaken using cows generated by somatic cell nuclear transfer (SCNT) from one founder, to produce a homogeneous population with reduced genetic and environmental variability. Oocytes and somatic tissues were collected from young (3 years of age; n = 4 females) and old (10 years of age; n = 5 females) cow clones following multiple natural ovarian cycles, as well as oocytes following multiple mild (FSH only) and standard (based on human a long GnRH agonist protocol) ovarian stimulation cycles. In addition, oocytes were recovered in a natural cycle from naturally conceived cows aged 4-13.5 years (n = 10) to provide a heterogeneous cohort for mtDNA deletion studies. The presence or absence of mtDNA deletions were investigated using long-range PCR in individual oocytes (n = 62). To determine the detection threshold for mtDNA heteroplasmy levels in individual oocytes, a novel NGS methodology was validated; artificial mixtures of the Bos taurus and Bos indicus mitochondrial genome were generated at 1, 2, 5, 15 and 50% ratios to experimentally mimic different levels of heteroplasmy. This NGS methodology was then employed to determine mtDNA heteroplasmy levels in single oocytes (n = 24). Oocyte mtDNA deletion and heteroplasmy data were analysed by binary logistic regression with respect to the effects of ovarian ageing and ovarian stimulation regimens. MAIN RESULTS AND THE ROLE OF CHANCE: Ovarian ageing, but not ovarian stimulation, increased the number of oocytes exhibiting mtDNA deletions (P = 0.04). A minimum mtDNA heteroplasmy level of 2% was validated as a sensitive (97-100%) threshold for variant detection in individual oocytes using NGS. Few mtDNA heteroplasmies were detected across the individual oocytes, with only 15 oocyte-specific variants confined to two of the 24 oocytes studied. There was no relationship (P > 0.05) evident between ovarian ageing or ovarian stimulation and the presence of mtDNA heteroplasmies. LIMITATIONS, REASON FOR CAUTION: The low number of oocytes collected from the natural ovarian cycles limited the analysis. Fertilization and developmental potential of the oocytes was not assessed as the oocytes were destroyed for mtDNA deletion and heteroplasmy analysis. WIDER IMPLICATIONS OF THE FINDINGS: If the findings of this model apply to the human, this study suggests that the incidence of mtDNA deletions increases with age, but not with degree of ovarian stimulation, while the frequency of mtDNA heteroplasmies may be low regardless of ovarian ageing or level of ovarian stimulation. STUDY FUNDING AND COMPETING INTERESTS: Funding was provided by Fertility Associates, the Nurture Foundation for Reproductive Research, the Fertility Society of Australia, and the Auckland Medical Research Foundation. J.C.P. is a shareholder of Fertility Associates and M.P.G. received a fellowship from Fertility Associates. The other authors of this manuscript declare no conflict of interest that could be perceived as prejudicing the impartiality of the reported research.

Assessing embryo quality by combining non-invasive markers: early time-lapse parameters reflect gene expression in associated cumulus cells.

STUDY QUESTION: Are there associations between early time-lapse parameters, expression of candidate embryo viability genes in cumulus cells and embryo quality on Day 5? SUMMARY ANSWER: Early time-lapse parameters correlate to the expression levels of candidate embryo viability genes in cumulus cells but a combined analysis including both time-lapse and candidate gene expression did not identify significant predictors of embryo quality on Day 5. WHAT IS KNOWN ALREADY: Recent evidence suggests that early time-lapse parameters are predictive of blastocyst development. Similarly, a number of candidate genes in cumulus cells have been identified as potential markers of embryo viability. Relationships between time-lapse parameters and candidate gene expression in cumulus cells have not been investigated, and a combined analysis of these markers has not been attempted in relation to embryo quality. STUDY DESIGN, SIZE, DURATION: A total of 78 embryos obtained by ICSI from 22 patients were studied by time-lapse and measurement of cumulus cell gene expression of known markers of embryo viability. Time-lapse and cumulus cell gene expression data were assessed in relation to embryo quality on Day 5. PARTICIPANTS/MATERIALS, SETTING, METHODS: All women, aged 32-40 years, underwent ICSI treatment for male infertility. Embryos with annotatable time to pronuclear breakdown (tPNB), division to two cells (t2C), three cells (t3C), four cells (t4C) and five cells (t5C) were included in the study. Expression levels of 27 candidate genes for embryo viability were measured in 78 associated cumulus cell masses using quantitative real-time PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Cumulus cell expression of 11 candidate genes involved in energy metabolism (ATPase, H+ transporting, lysosomal 70 kDa, V1 subunit A (ATP6V1A), NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 1, 7.5 kDa (NDUFA1), lactate dehydrogenase A (LDHA), phosphofructokinase platelet (PFKP) and solute carrier family 2 member 4 (SLC2A4), mitochondrial biogenesis (DNA directed RNA polymerase, mitochondrial (POLRMT) and transcription factor A, mitochondrial (TFAM), signalling (prostaglandin-endoperoxide synthase 2), steroidogenesis (cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) and cell stress (heat shock 70 kDa protein 5 (HSPA5) and peroxiredoxin 3 (PRDX3)) correlated to time-lapse parameters of the developing embryo, largely for t3C onwards (all P < 0.05). Expression of ATP synthase, H+ transporting, mitochondrial Fo complex, subunit E (ATP51), HSPA5, PFKP, PRDX3 and versican (VCAN) and the parameter t4C were also related to embryo quality on Day 5 (all P < 0.05). Ordinal logistic regression, where gene expression and time-lapse parameters were combined, did not identify any significant predictors of embryo quality on Day 5. LIMITATIONS AND REASON FOR CAUTION: Data are from a preliminary study, limited by a small sample size and using more than one ovarian stimulation protocol. A possible limitation is that each follicle was treated as an independent observation, although a considerable fraction of embryos were from the same patient. WIDER IMPLICATIONS OF THE FINDINGS: Results presented in this study suggest that some of the variation of time-lapse parameters may be related to cumulus cell gene expression and thus the ovarian microenvironment in which the oocyte developed. Although the current study did not identify significant predictors of embryo quality on Day 5, investigation in a larger cohort may determine whether cumulus cell gene expression and time-lapse parameters can be combined to predict embryo quality. STUDY FUNDING/COMPETING INTERESTS: Funding was provided by Fertility Associates Ltd, the Auckland Medical Research Foundation and the University of Auckland. J.C.P. has a 0.5% shareholding in Fertility Associates. All other authors of this manuscript have nothing to declare and no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Many women undergoing fertility treatment make poor lifestyle choices that may affect treatment outcome.

STUDY QUESTION: What are the lifestyle choices and dietary aspects of women about to undergo fertility treatment in New Zealand? SUMMARY ANSWER: A considerable proportion of women about to undergo fertility treatment make poor lifestyle choices, including the consumption of alcohol and caffeine. WHAT IS KNOWN ALREADY: Women undergoing fertility treatment are highly motivated to achieve pregnancy, but there are relatively few published data on their lifestyle, lifestyle changes or dietary aspects. STUDY DESIGN, SIZE, DURATION: This was a cross-sectional study of 250 women aged 20-43 years, taking place between March 2010 and August 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women about to undergo IVF or ICSI treatment in two large fertility clinics in Auckland and Hamilton, New Zealand. Lifestyle and dietary intake questionnaires were individually administered once to each participant 35 days (SD = 22) prior to fertility treatment initiation. Outcome measures included incidence of smoking, consumption of alcohol and caffeinated beverages, BMI, detailed intake of dietary supplements and fertility treatment success. Consumption of certain nutrient supplements was compared with the general female New Zealand population. MAIN RESULTS AND THE ROLE OF CHANCE: There were high rates of alcohol (50.8%) and caffeine (86.8%) consumption. Most women (82.8%) reported at least one lifestyle change in preparation for fertility treatment, but less than half of women who consumed alcohol regularly reduced their intake and 60% did not change consumption of caffeinated beverages. Similarly, the majority of women did not change their exercise levels (64.4%) or BMI (83.6%) ahead of fertility treatment. Coffee intake appeared unrelated to treatment outcome, but women who consumed caffeinated herbal tea (36.4% of the study population consumed green tea) had lower odds of becoming pregnant (odds ratio, OR 0.52; P = 0.041 versus those not consuming caffeinated herbal tea). Women who abstained from drinking or reduced alcohol intake had twice the odds of becoming pregnant than those who maintained their drinking habits prior to fertility treatment (OR 2.27; P = 0.049). While 93.2% of women took a folic acid supplement, 16.8% had an inadequate intake compared with the current New Zealand prenatal recommendation of 800 mcg/day. Women who held a university degree or higher qualification had twice the odds of becoming pregnant as women with lower levels of education (OR 2.08; P = 0.017), though this finding appeared to be unrelated to lifestyle or dietary habits. LIMITATIONS, REASONS FOR CAUTION: The study involved self-reported behaviours that might have been misrepresented by respondents. In addition, our questionnaires covered the period following the first clinical assessment but ∼5 weeks prior to fertility treatment initiation, so that we cannot ascertain whether dietary intakes and lifestyle choices persisted over the course of treatment itself. WIDER IMPLICATIONS OF THE FINDINGS: Many women about to undergo fertility treatment make poor lifestyle choices that may negatively affect their chances of becoming pregnant. These findings may be more widely applicable to other women attempting to become pregnant. Specific advice for women regarding healthy lifestyle choices while undergoing fertility treatment is warranted. STUDY FUNDING/COMPETING INTERESTS: A.A.G. received financial support from Abbott Nutrition Research & Development Asia-Pacific Center; J.C.P. is a shareholder of Fertility Associates; the other authors have no financial or non-financial conflicts of interest to disclose.

Maternal age and ovarian stimulation independently affect oocyte mtDNA copy number and cumulus cell gene expression in bovine clones.

STUDY QUESTION: Does maternal ageing and ovarian stimulation alter mitochondrial DNA (mtDNA) copy number and gene expression of oocytes and cumulus cells from a novel bovine model for human IVF? SUMMARY ANSWER: Oocytes collected from females with identical nuclear genetics show decreased mtDNA copy number and increased expression of an endoplasmic reticulum (ER) stress gene with repect to ovarian stimulation, whilst differences in the expression of genes involved in mitochondrial function, antioxidant protection and apoptosis were evident in relation to maternal ageing and the degree of ovarian stimulation in cumulus cells. WHAT IS KNOWN ALREADY: Oocyte quality declines with advancing maternal age; however, the underlying mechanism, as well as the effects of ovarian stimulation are poorly understood. Human studies investigating these effects are often limited by differences in age and ovarian stimulation regimens within a patient cohort, as well as genetic and environmental variability. STUDY DESIGN, SIZE, DURATION: A novel bovine cross-sectional maternal age model for human IVF was undertaken. Follicles were aspirated from young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian clones following multiple unstimulated, mild and standard ovarian stimulation cycles. These bovine cloned females were generated by the process of somatic cell nuclear transfer (SCNT) from the same founder and represent a homogeneous population with reduced genetic and environmental variability. Maternal age and ovarian stimulation effects were investigated in relation to mtDNA copy number, and the expression of 19 genes involved in mitochondrial function, antioxidant protection, oocyte-cumulus cell signalling and follicle development in both oocytes and cumulus cells. MATERIALS, SETTING, METHODS: Young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian bovine clones were maintained as one herd. Stimulation cycles were based on the long GnRH agonist down-regulation regimen used in human fertility clinics. Follicle growth rates, numbers and diameters were monitored by ultrasonography and aspirated when the lead follicles were >14 mm in diameter. Follicle characteristics were analysed using a mixed model procedure. Quantitative PCR (qPCR) was used to determine mtDNA copy number and reverse transcriptase-qPCR (RT-qPCR) was used to measure gene expression in oocytes and cumulus cells. MAIN RESULTS AND THE ROLE OF CHANCE: Method of ovarian stimulation (P = 0.04), but not maternal age (P > 0.1), was associated with a lower mtDNA copy number in oocytes. Neither factor affected mtDNA copy number in cumulus cells. In oocytes, maternal age had no effect on gene expression; however, ovarian stimulation in older females increased the expression of GRP78 (P = 0.02), a gene involved in ER stress. In cumulus cells, increasing maternal age was associated with the higher expression of genes involved in mitochondrial maintenance (TXN2 P = 0.008 and TFAM P = 0.03), whereas ovarian stimulation decreased the expression of genes involved in mitochondrial oxidative stress and apoptosis (TXN2 P = 0.002, PRDX3 P = 0.03 and BAX P = 0.03). LIMITATIONS, REASON FOR CAUTION: The low number of oocyte and cumulus cell samples collected from the unstimulated cycles limited the analysis. Fertilization and developmental potential of the oocytes was not assessed because these were used for mtDNA and gene expression quantification. WIDER IMPLICATIONS OF THE FINDINGS: Delineation of the independent effects of maternal age and ovarian stimulation regimen on mtDNA copy number gene expression in oocytes and cumulus cells was enabled by the removal of genetic and environmental variability in this bovine model for human IVF. Therefore, these extend upon previous knowledge and findings provide relevant insights that are applicable for improving human ovarian stimulation regimens. STUDY FUNDING/COMPETING INTERESTS: Funding was provided by Fertility Associates and the University of Auckland. J.C.P. is a shareholder of Fertility Associates and M.P.G. received a fellowship from Fertility Associates. The other authors of this manuscript declare no conflict of interest that could be perceived as prejudicing the impartiality of the reported research.

Phenotypic differences in children conceived from fresh and thawed embryos in in vitro fertilization compared with naturally conceived children.

OBJECTIVE: To determine whether anthropometric and biochemical features differ in in vitro fertilization (IVF) children conceived via fresh (IVFF) or thawed (IVFT) embryo transfer compared with naturally conceived controls. DESIGN: A cross-sectional controlled study. SETTING: University clinical research unit. PATIENT(S): Healthy prepubertal children (3.5-11.0 years), singletons, born at term (>37 weeks), who were either naturally conceived (controls; n = 94) or IVF children conceived via the transfer of a fresh (IVFF; n = 72) or thawed (IVFT; n = 43) embryo. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Assessments of anthropometry (adjusted for parental variables), dual-energy X-ray absorptiometry-derived body composition, fasting plasma growth factors, lipids, and parameters of glucose regulation. RESULT(S): The IVFF but not the IVFT children weighed less at birth than the control children. The IVFF children were taller than both the controls and IVFT children. Sex-specific analyses showed height differences among girls, with IVFF girls being taller than their control and IVFT counterparts. Taller stature in IVFF children was associated with increased insulin-like growth factor I (IGF-I) concentrations compared with controls, whereas the IVFT children displayed increased IGF-II and decreased insulin-like growth factor binding protein 3 (IGFBP-3) concentrations compared with the controls. More favorable lipid profiles were also evident in IVFF but not IVFT children compared with the control children. CONCLUSION(S): These preliminary findings highlight that the transfer of a fresh versus a thawed IVF embryo affects height, plasma growth factor, and lipid profiles in childhood. Therefore, embryo derivation should be considered when assessing physical and biochemical phenotype of IVF children.

Ovarian stimulation leads to shorter stature in childhood.

BACKGROUND: We aimed to determine whether children conceived with ovarian stimulation alone (OS(A)) would differ phenotypically and biochemically from naturally conceived children of fertile and subfertile parents. METHODS: Healthy pre-pubertal children aged 3-10 years, born at term, after singleton pregnancies were recruited in Auckland (New Zealand) and were allocated into three groups: (i) children conceived following OS(A) and naturally conceived children of (ii) subfertile and (iii) fertile parents. Anthropometric, endocrine and metabolic parameters were recorded. Children's heights and body mass index (BMI) were expressed as standard deviation scores (SDS) and corrected for genetic potential (i.e. parental height or BMI). RESULTS: Three hundred fifty-two children were studied: 84 OS(A) subjects and 268 naturally conceived controls consisting of 54 children of subfertile parents and 214 children of fertile parents. Children of subfertile and fertile parents did not differ in measured outcomes. Overall, OS(A) children were shorter than children of both subfertile (SDS: -0.08 ± 0.09 versus 0.32 ± 0.07; P= 0.001) and fertile (SDS: -0.08 ± 0.09 versus 0.45 ± 0.10; P= 0.004) parents when corrected for genetic height potential. OS(A) boys were shorter than boys of subfertile (SDS:-0.18 ± 0.14 versus 0.42 ± 0.16; P= 0.03) and fertile (SDS: -0.18 ± 0.14 versus 0.35 ± 0.08; P= 0.01) parents. There was also a trend towards OS(A) girls being shorter than girls of subfertile parents (P= 0.06), but not significantly shorter than those of fertile parents (P= 0.17). OS(A) children also had a lower corrected BMISDS than children of subfertile (SDS-0.90 ± 0.15 versus -0.37 ± 0.17; P= 0.06) and fertile (-0.90 ± 0.15 versus -0.34 ± 0.10; P= 0.008) parents. Among metabolic parameters, fasting glucose was lower in OS(A) children than that in children of fertile parents (4.62 ± 0.07 versus 4.81 ± 0.04; P= 0.006). CONCLUSIONS: Conception after OS(A) was associated with shorter stature, particularly in boys, compared with naturally conceived children of fertile and subfertile parents.

Development of clinical priority access criteria for assisted reproduction and its evaluation on 1386 infertile couples in New Zealand.

BACKGROUND: In New Zealand ranking patients for elective, publicly funded procedures uses clinical priority access criteria (CPAC). A CPAC to prioritize patients seeking assisted reproductive technology (ART) was developed in 1997 and implemented nationwide in 2000. This study describes the development of the ART CPAC tool and its evaluation on 1386 couples referred to a single tertiary service from 1998 to 2005. METHODS: A total of 48 health professionals and consumers assisted in criteria development. A score between 0 and 100 points was calculated for each couple and those who reached ≥65 points were eligible for publicly funded ART. Couples beneath the treatment threshold were placed on active review; the review being the date the score was calculated to reach the treatment threshold. Couples who would never be eligible or who were on active review were offered private treatment. Treatments and outcomes (spontaneous and treatment dependent live birth pregnancies) were used to evaluate the criteria. RESULTS: Three social criteria (duration infertility, number of children and sterilization status) and two objective criteria (diagnosis and female age) formed the priority score. Of the evaluated couples, 643 (46%) were eligible within 1 year of referral (Group 1), 451 (33%) >1-5 years from referral (Group 2) and 292 (21%) couples were never eligible (Group 3). The predominant ART was IVF. A total of 480 couples had at least one IVF treatment with 404 (84%) having publicly funded treatment. A total of 762 (55%) women gave birth, 473 from treatment and 289 spontaneously. Group 1 had more pregnancies from treatment while Group 2 had most pregnancies overall being mainly from spontaneous pregnancies. Compared with Group 3 cases the hazard ratio using time to spontaneous live birth pregnancy for Group 1 couples was significantly lower, 0.51 (95% confidence interval 0.36-0.74) and for Group 2 cases significantly higher, 1.86, (1.35-2.58). Treatments using ART were evaluated for the three eligibility groups, with the never eligible divided into women age <40 (Group 3a) and woman age ≥40 at referral (Group 3b). Compared with Group 1 cases the hazard ratio to treatment dependent live birth pregnancy was similar for Groups 2 and 3a but significantly lower for Group 3b (0.37, 0.14-0.90). CONCLUSIONS: The clinical priority access score was able to discriminate between the chance of pregnancy with and without treatment and those offered and not offered treatment. The CPAC is a useful model for informing the allocation of public funding for ART in other countries.