Mark Bowman

What next for preimplantation genetic screening (PGS)? Experience with blastocyst biopsy and testing for aneuploidy

Blastocysts more commonly have a normal karyotype than cleavage-stage embryos do. Moreover, blastocysts have also made a metabolic transition from catabolism and recycling of the oocytes reserves and resources, processes that fuel the first 3 days of cleavage. Although not all blastocysts are karyotypically equal, it is still to be determined to what extent a mosaic karyotype might be a normal feature among embryos, both at the cleavage stage and the blastocyst stage--and when looking for karyotypic abnormalities by embryo biopsy might help the chance of implantation rather than harm it. It is also still impractical to look at all the chromosomes that can, through their aneuploidy, stand in the way of successful embryonic and fetal development. We report a randomized clinical trial of blastocyst biopsy followed by preimplantation genetic screening (PGS) for aneuploidy using 5-colour FISH. The trial was suspended and then terminated early when we were unable to show an advantage for PGS. If we are correct in assuming that mitotic non-disjunction is common by the stage of the blastocyst (and that it is much less ominous than meiotic non-disjunction), then further studies of effective PGS of blastocysts for aneuploidy require methods of analysis that cover all the chromosomes and can differentiate the triallelic and monoallelic states of meiotically derived aneuploidies from the biallelic state of mitotic aneuploidies.

Single-embryo transfer of vitrified-warmed blastocysts yields equivalent live-birth rates and improved neonatal outcomes compared with fresh transfers

OBJECTIVE To compare pregnancy and neonatal outcomes after fresh and vitrified-warmed single-blastocyst transfers. DESIGN Retrospective study. SETTING Private in vitro fertilization (IVF) clinic. PATIENT(S) 1,209 infertile patients who underwent a total of 1,157 fresh and 645 vitrified-warmed embryo transfers. INTERVENTION(S) Day-5 single-blastocyst transfers using fresh or vitrified-warmed (Cryotop method) grade I and grade II embryos. MAIN OUTCOME MEASURE(S) Fetal heart pregnancy rate, live-birth rate, gestational age, and live-birth weight. RESULT(S) The overall blastocyst thaw survival rate was 94.4% and was not significantly different between blastocyst grades or developmental stages. Similar clinical outcomes were achieved for fresh and vitrified-warmed blastocyst transfers; for example, grade I blastocysts had a live-birth rate of 52.8% versus 55.3%, respectively, and grade II blastocysts had a rate of 34.9% versus 30.4%, respectively. Significantly improved neonatal outcomes were evident for vitrified-warmed blastocyst transfers for gestational age, being on average 0.3 weeks longer, and for live-birth weight with babies born on average 145 g heavier (3,296 g versus 3,441 g for fresh and vitrified-warmed groups, respectively), as compared with fresh transfers. CONCLUSION(S) Embryo transfer of vitrified-warmed blastocysts yields equivalent live-birth rates and improved neonatal outcomes compared with fresh transfers.

Do Alterations in the Sex Ratio Occur at Fertilization? A Case Report Using Fluorescent In Situ Hybridization

Purpose:The mechanisms by which the sex ratio might be altered at fertilization were reviewed, following a case of preimplantation gender analysis revealing a significantly skewed proportion of male-to-female embryos.Methods:The case of a known carrier of X-linked hydrocephalus with a history of three affected male pregnancies is presented. Her husbands family history consisted of a strong increase in the number of males relative to females. She had four cycles of stimulated in vitro fertilization, with sex chromosome analysis using fluorescent in situ hybridization (FISH) on suitable cleavage-stage embryos. The difference in the sex ratio of normal male-to-female embryos was compared using a significance probabilities test for sex ratio. The sex ratio of sperm from a semen sample from the male partner was determined by FISH.Results:Fifty embryos were suitable for analysis. A significantly higher number of normal male (n = 20) than normal female (n = 8) embryos was obtained (P < 0.05). The FISH assessment of the husbands semen analysis revealed no alteration in the normal X:Y ratio.Conclusions:As the sperm analysis revealed a normal X:Y ratio, an alteration in the embryo sex ratio might be explained by the preferential binding of Y-bearing sperm to the oocyte, an oocyte-related “discouragement” of binding of X-bearing sperm, or a postfertilization event.

Gestational surrogacy in Australia 2004‐2011: treatment, pregnancy and birth outcomes

Information on gestational surrogacy arrangement and outcomes is limited in Australia.

Socio-economic disparities in access to assisted reproductive technologies in Australia

Women from disadvantaged socio-economic groups access assisted reproductive technology treatment less than women from more advantaged groups. However, women from disadvantaged groups tend to start families younger, making them less likely to suffer from age-related subfertility and potentially have less need for fertility treatment. Whether socio-economic disparities in access to assisted reproductive technology treatment persist after controlling for the need for treatment, has not been previously explored. This population based study demonstrates that socio-economic disparities in access to assisted reproductive technology treatment persist after adjusting for several confounding factors, including age at first birth (used as a measure of delayed childbearing, hence a proxy for need for fertility treatment), geographic remoteness and Australian jurisdiction. Assisted reproductive technology access progressively decreased as socio-economic quintiles became more disadvantaged, with a 15.8% decrease in access in the most disadvantaged quintile compared with the most advantaged quintile after controlling for confounding factors. The adjusted rate of access to assisted reproductive technology treatment also decreased by 12.3% for women living in regional and remote areas compared with those in major cities. These findings indicate that financial and sociocultural barriers to assisted reproductive technology treatment remain in disadvantaged groups after adjusting for need.

Use of the CryoPredict algorithm to predict live birth from cryopreserved embryos.

Currently, the viability of cryostored blastocysts that are subsequently re‐warmed is determined via the percentage of cell survival. However, the large number of cells that forms the blastocyst can make this estimate difficult and unreliable. Studies have shown that fast re‐expanding blastocysts have superior pregnancy rates.

Comment on “MeDIP real‐time qPCR of maternal peripheral blood reliably identifies trisomy 21”

We read with interest the recent publication in your journal by Tsaliki et al. titled ‘MeDIP real-time qPCR of maternal peripheral blood reliably identifies trisomy 21’. In a correspondence published in September issue of Nature Medicine, Tong et al. raised technical concerns about this methodology. Experimentally, Tong et al. failed to reproduce the results using 15 normal and 3 trisomy cases. We wish to communicate our preliminary results with colleagues working in this field. We used cell-free circulating DNA extracted from 1.5 to 2mL ofmaternal plasma fromwomen during the first trimester of pregnancy as the starting material. Following the enrichment of methylated DNA using a methodology to be published later by our group, real time Q-PCR was performed using eight molecular markers, EP4–8 and EP10–12, as shown in experiments by Papageorgious and Tong et al. Thirty normal samples and two trisomy 21 cases were examined. We calculated methylation ratios that showed similar ranges for each marker to those presented by Papageorgious et al. We tentatively used the equation published by Papageorgious to calculateD values.Wewere able to correctly identify both trisomy cases and 29 normal cases, although one normal case had a D value of 0.65. We will be able to test the data more accurately once our own equation is developed. We believe that statistically, the equation obtained from work by Papageorgious et al. may only represent the data obtained from their experimental conditions and reagents used. The coefficients in the equation are not necessarily accurate for the data collected in other laboratories unless these coefficients are determined under a standardized system and from a large enough sample size that is statistically able to represent general population of women with first trimester pregnancy. Therefore, we believe that it would be prudent to be cautious when using the equation to explain another set of data. We are not surprised to see that D values varied for the replicates in the reproducibility experiments of Tong et al. Once the coefficients are determined on the basis of a particular set of data, the methylation ratio is an independent variable that ultimately determines theD value. In this context, themethylation ratio is defined as normalized delta Ct for an individual sample against the median normalized delta Ct of a group of known normal samples. In a quantitative polymerase chain reaction (PCR), variation in Ct values amongmultiple replicates of a sample is generally acceptedwithin the half cycle. This allowable, less than half-cycle, difference can produce a range of variation in the D value for any sample given that eight markers contribute to the formation of the D value in this equation. Existence of this variation is logical as long as it does not change the nature of the tested sample that, in this scenario, is trisomy 21 or euploidy. We also tested the reproducibility of methylation enrichment. Within a run, five technical replicates of a single sample were examined. Ct values for five enriched samples were all centered on a value with less than half-cycle difference. With runs on different days, we were able to reach 99–100% consistency for the readings of Ct values when comparing twomeans from each run. In terms of efficiency of enrichment, reproducibility between runs for the eight markers examined is as follows: EP4, 99.30%; EP5, 93.65%; EP6, 98.90%; EP7, 91.14; EP8, 88.89%, EP10, 100%; EP11, 86.96; and EP12, 88.52%. Furthermore, we examined the reproducibility of methylation enrichment using representative markers EP4 and EP5 within a period of 1month, and we are able to reproduce five weekly results with variations of less than half a PCR cycle. Our preliminary results suggest that it is worthwhile to further validate this methodology in a large sample cohort.

Linking Sleep Disturbance To Idiopathic Male Infertility

Recently published data suggests that male fertility has declined over the past four decades. The reasons for the decline are unclear with up to 50% of cases of male infertility remaining unexplained (idiopathic male infertility). Whilst environmental factors and rising rates of obesity have been implicated, there is now growing evidence that sleep disturbance may be an independent causative factor. Indeed, the prevalence of sleep disturbance appears to be increasing in parallel with deterioration in population sperm quality, a commonly used surrogate marker of male fertility. Although there is some understanding of the relationship between sleep, gonadal hormone secretion and sexual function, it remains to be seen whether sleep disturbance is implicated in idiopathic male infertility. This review will detail the current evidence supporting a link between sleep disturbance and male infertility. Potential mechanistic pathways will be proposed and evidence supporting these pathways will be discussed. Further research is needed in clarifying links between sleep disturbance and idiopathic male infertility. At present the only available treatment option for men with idiopathic infertility is assisted reproductive technology. Demonstration of a causative link between sleep disturbance and idiopathic male infertility may in the future lead to additional treatment options in selected cases.

Continuous improvement in national ART standards by the RTAC accreditation system in Australia and New Zealand

Assisted reproductive technology (ART) clinics in Australia and New Zealand are accredited and licensed against a Code of Practice audited by certifying bodies accredited by the Joint Accreditation System for Australia and New Zealand (JAS‐ANZ). The system is administered by the Reproductive Technology Accreditation Committee (RTAC) of the Fertility Society of Australia.