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

BACKGROUND: 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. AIM: To determine whether the degree and speed of blastocoele re-expansion following cryopreservation and warming correlate with rates of live birth. MATERIALS AND METHODS: A retrospective cohort study of 757 frozen embryo transfer cycles over a 4-year period at Royal Prince Alfred Hospital, Sydney. Clinical and embryology notes were retrieved. Details regarding patient demographics, stimulation cycle from which embryos were derived, frozen embryo transfer cycles, embryology and pregnancy outcomes were recorded. RESULTS: Female (P = 0.01) and male age (P = 0.02) at the time of embryo creation were inversely associated with live birth. Fertilisation method (P = 0.03), embryo type at cryopreservation (P = 0.009), embryo grade at cryopreservation (P < 0.0001), percentage of cell survival post-thaw (P < 0.0001) and the degree of re-expansion (P = 0.003) were the IVF and embryology factors significantly associated with live birth. A predictive model (CryoPredict) was created in order to individualise the probability that the transfer of a given embryo would result in live birth. CONCLUSIONS: The degree and speed of blastocoele re-expansion postcryopreservation and subsequent warming can be used in conjunction with other parameters to predict live birth.

The original Beckman Coulter Generation II assay significantly underestimates AMH levels compared with the revised protocol.

PURPOSE: Anti-Müllerian hormone (AMH) is used as a marker for ovarian reserve. Since 2011, the standard test for AMH has been the Beckman Coulter Generation (Gen) II assay. However, in July 2013, the protocol was revised due to falsely low readings. The aim of this study was to compare AMH levels measured with the original and revised Gen II assay and to establish a fertile female reference range for the revised protocol. METHODS: Serum AMH levels were measured for 492 natural conception first trimester pregnant women using the original and revised Gen II assay. RESULTS: The original protocol significantly underestimated AMH levels compared with the revised protocol (p < 0.001), the median being 8.4 and 14.2 pmol/L, respectively. In all samples with detectable AMH levels, the revised protocol yielded a higher concentration compared with the original protocol, the magnitude shift ranging from 3.4 to 283.3 % (median 68.0 %). AMH levels measured with the revised protocol were collated to generate an age-specific reference range, with median levels peaking at 27 years then declining with advancing age. The median AMH concentration for ages 20-24 was 17.3 pmol/L, ages 25-29 was 20.5 pmol/L, ages 30-34 was 17.8 pmol/L, ages 35-39 was 10.8 pmol/L, and ages 40-44 was 6.1 pmol/L. CONCLUSIONS: Our study demonstrated that the original Gen II assay significantly underestimated AMH levels, suggesting caution is required when interpreting literature and testing results achieved with this assay. We also established the revised Gen II assay reference range for AMH in women with unassisted proven fertility.

Embryo vitrification using a novel semi-automated closed system yields in vitro outcomes equivalent to the manual Cryotop method.

STUDY QUESTION: Can the equilibration steps prior to embryo vitrification be automated? SUMMARY ANSWER: We have developed the 'Gavi' system which automatically performs equilibration steps before closed system vitrification on up to four embryos at a time and gives in vitro outcomes equivalent to the manual Cryotop method. WHAT IS KNOWN ALREADY: Embryo cryopreservation is an essential component of a successful assisted reproduction clinic, with vitrification providing excellent embryo survival and pregnancy outcomes. However, vitrification is a manual, labour-intensive and highly skilled procedure, and results can vary between embryologists and clinics. A closed system whereby the embryo does not come in direct contact with liquid nitrogen is preferred by many clinics and is a regulatory requirement in some countries. STUDY DESIGN, SIZE, DURATION: The Gavi system, an automation instrument with a novel closed system device, was used to equilibrate embryos prior to vitrification. Outcomes for embryos automatically processed with the Gavi system were compared with those processed with the manual Cryotop method and with fresh (non-vitrified) controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: The efficacy of the Gavi system (Alpha model) was assessed for mouse (Quackenbush Swiss and F1 C57BL/6J x CBA) zygotes, cleavage stage embryos and blastocysts, and for donated human vitrified-warmed blastocysts. The main outcomes assessed included recovery, survival and in vitro embryo development after vitrification-warming. Cooling and warming rates were measured using a thermocouple probe. MAIN RESULTS AND THE ROLE OF CHANCE: Mouse embryos vitrified after processing with the automated Gavi system achieved equivalent in vitro outcomes to that of Cryotop controls. For example, for mouse blastocysts both the Gavi system (n = 176) and manual Cryotop method (n = 172) gave a 99% recovery rate, of which 54 and 50%, respectively, progressed to fully hatched blastocysts 48 h after warming. The outcomes for human blastocysts processed with the Gavi system (n = 23) were also equivalent to Cryotop controls (n = 13) including 100% recovery for both groups, of which 17 and 15%, respectively, progressed to fully hatched blastocysts 48 h after warming. The cooling and warming rates achieved with the Gavi system were 14 136°C/min and 11 239°C/min, respectively. LIMITATIONS, REASONS FOR CAUTION: Testing of the Gavi system described here was limited to in vitro development of embryos from two mouse strains and a limited number of human embryos. Validation of Gavi system advanced production models is now required to confirm the success of semi-automated vitrification, including clinical evaluation of pregnancy outcomes from the transfer of Gavi vitrified-warmed human embryos. WIDER IMPLICATIONS OF THE FINDINGS: The Gavi system has the potential to revolutionize and standardize vitrification of embryos and oocytes. The success of the Gavi system shows that it is possible to semi-automate complicated labour-intensive ART methods and processes, and opens up the possibility for further improvements in clinical outcomes and efficiencies in the ART clinic. STUDY FUNDING/COMPETING INTERESTS: This study was funded by Genea Ltd. S.B., N.M.T., T.T.P., S.J.M., M.C.B. and T.S. are shareholders of Genea Ltd. E.V., C.H., C.L., S.R.L. and S.M.D. are shareholders of Planet Innovation Pty Ltd. The remaining authors are employees of either Genea Ltd. or Planet Innovation Pty Ltd.

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 oocyte's 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.

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.

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.

Elective transfer of single fresh blastocysts and later transfer of cryostored blastocysts reduces the twin pregnancy rate and can improve the in vitro fertilization live birth rate in younger women.

OBJECTIVE: To determine the extent to which embryo selection by blastulation and elective single blastocyst transfer, supported by efficient cryostorage of spare embryos, can reduce multiple pregnancies and maintain or improve on the live birth rate from IVF. DESIGN: Prospective, nonrandomized cohort study. SETTING: Sydney IVF, a private clinic in Australia. PATIENT(S): In vitro fertilization patients aged <38 years with three or more usable blastocyst, recruited from April 2000 through December 2001, with pregnancies followed up until August 2004. INTERVENTION(S): Blastocysts were cultured and cryostored with stage-specific culture medium and low oxygen conditions. MAIN OUTCOME MEASURE(S): Fetal heart-positive twin pregnancy rate and accumulating "take-home baby" rate per retrieval leading to blastocyst transfer. RESULT(S): Among 121 women who elected single fresh blastocyst transfer (but who could elect to have two frozen blastocysts transferred at once), 79 (65.3%) took home a baby, with a twin pregnancy rate of 7%. Among 285 women who chose two blastocysts for fresh transfer, 184 (64.2%) took home at least one baby, with a twin pregnancy rate of 34% and five perinatal deaths. CONCLUSION(S): With technically appropriate blastocyst culture and freezing, and elective single blastocyst transfer in the fresh cycle, the overall multiple pregnancy rate can be reduced by >75%, permitting in this series a slight increase in the chance of taking home a baby.