D.H. McCulloh

Diminished effect of maternal age on implantation after preimplantation genetic diagnosis with array comparative genomic hybridization.

OBJECTIVE To assess the relationship between maternal age, chromosome abnormality, implantation, and pregnancy loss. DESIGN Multicenter retrospective study. SETTING IVF centers in the United States. PATIENT(S) IVF patients undergoing chromosome screening. INTERVENTION(S) Embryo biopsy on day 3 or day 5/6 with preimplantation genetic diagnosis (PGD) by array comparative genomic hybridization. MAIN OUTCOME MEASURE(S) Aneuploidy, implantation, pregnancy, and loss rates. RESULT(S) Aneuploidy rates increased with maternal age from 53% to 93% for day 3 biopsies and from 32% to 85% for blastocyst biopsies. Implantation rates for euploid embryos for ages <35-42 years did not decrease after PGD: ranges 44%-32% for day 3 and 51%-40% for blastocyst. Ongoing pregnancy rates per transfer did not decrease for maternal ages <42 years after PGD with day 3 biopsy (48.5%-38.1%) or blastocyst biopsy (64.4%-54.5%). Patients >42 years old had implantation rates of 23.3% (day 3), 27.7% (day 5/6), and the pregnancy rate with day 3 biopsy was 9.3% and with day 5 biopsy 10.3%. CONCLUSION(S) Selective transfer of euploid embryos showed that implantation and pregnancy rates were not significantly different between reproductively younger and older patients up to age 42 years. Some patients who start an IVF cycle planning to have chromosome screening do not have euploid embryos available for transfer, a situation that increases with advancing maternal age. Mounting data suggests that the dramatic decline in IVF treatment success rates with female age is primarily caused by aneuploidy.

Correlative ultrastructural and electrophysiological studies of sperm-egg interactions of the sea urchin, Lytechinus variegatus☆

The sequence of ultrastructural events following the onset of the sperm-induced conductance increase in eggs of the sea urchin, Lytechinus variegatus, was investigated. Eggs voltage clamped at -20 mV were fixed 1 to 20 sec after onset of the conductance increase caused by single sperm. Continuity between the plasma membranes of the sperm and egg was first detected 5 sec after onset of the conductance increase. The earliest stages of formation of the fertilization cone coincided with the establishment of continuity of the gamete plasma membranes. At 6 to 8 sec after the initial conductance increase cortical granule dehiscence was first observed in the immediate vicinity where continuity of the gamete plasma membranes had occurred. These observations are consistent with the conclusion that opening of ion channels at fertilization precedes fusion of the sperm and egg plasma membranes, while exocytosis of cortical granules is initiated following fusion of the sperm and egg plasma membranes.

Voltage clamp studies of fertilization in sea urchin eggs: II. Current patterns in relation to sperm entry, nonentry, and activation

Following attachment of a sperm to the surface of a sea urchin egg clamped at a membrane potential (Vm) more positive than +17 mV, no changes in membrane conductance can be detected, the sperm does not enter egg, and no morphological changes can be detected. At Vm from +17 to -100 mV three characteristically different types of current profiles are observed: Type I are activation currents in eggs penetrated by a sperm. These have three phases, which occur in all eggs clamped at Vm from +17 to -20 mV and in decreasing percentages at clamped Vm more negative than -20 mV (to -75 mV). Complete fertilization envelopes are elevated, relatively large mound-shaped fertilization cones form, and the eggs develop to normal embryos. Type II are sperm transient currents in eggs not penetrated by a sperm, the eggs otherwise remaining in the unfertilized state. These transients are simpler and shorter than type I currents, and are observed only at clamped Vm more negative than -20 mV. Type III are modified activation currents in eggs not penetrated by a sperm. These have three phases, are observed only at clamped Vm more negative than -20 mV, and are the only type of activation current seen at clamped Vm more negative than -75 mV. Complete fertilization envelopes are elevated, the fertilization cones are small and filament-like, and the eggs fail to cleave. We conclude that (a) the sperm transient currents (type II) and phase 1 of the activation currents (type I and III) are similar events generated by a sperm-initiated localized conductance increase, (b) the abrupt decrease of current which terminates the sperm transients and phase 1 of type III currents results from a turnoff of the sperm-induced conductance increase and signals that the sperm will not enter the egg, and (c) the occurrence of phase 2 during an electrophysiological response induced by a sperm indicates that the egg is activating.

Blastocyst culture selects for euploid embryos: comparison of blastomere and trophectoderm biopsies

Preimplantation genetic diagnosis and screening improves the chances of achieving a viable pregnancy, not only free of undesired single-gene defects but also aneuploidy. In addition, improvements in vitrification provide an efficient means of preserving embryos (blastocysts). By combining trophectoderm biopsy with recent improvements in vitrification methods, only those embryos that have proved themselves viable and potentially more competent are tested. Using array comparative genomic hybridization (aCGH) to assess all 24 chromosomes, aneuploidy rates were compared between day-3 blastomere biopsy and day-5 trophectoderm biopsy. Of those 1603 embryos, 31% were euploid, 62% were aneuploid and 7% not analysable. A significantly larger proportion of embryos were euploid on day-5 biopsy (42%) compared with day-3 biopsy (24%, P<0.0001). The number of euploid embryos per patient was not significantly different. Combining extended culture, trophectoderm biopsy and aneuploidy assessment by aCGH and subsequent vitrification can provide a more efficient means of achieving euploid pregnancies in IVF.

Why do euploid embryos miscarry? A case-control study comparing the rate of aneuploidy within presumed euploid embryos that resulted in miscarriage or live birth using next-generation sequencing

OBJECTIVE To determine whether undetected aneuploidy contributes to pregnancy loss after transfer of euploid embryos that have undergone array comparative genomic hybridization (aCGH). DESIGN Case-control study. SETTING University-based fertility center. PATIENT(S) Cases included 38 patients who underwent frozen euploid ET as determined by aCGH, resulting in miscarriage. Controls included 38 patients who underwent frozen euploid ET as determined by aCGH, resulting in a live birth. INTERVENTION(S) Next-generation sequencing (NGS) protocols were internally validated. Saved amplified DNA samples from the blastocyst trophectoderm biopsies previously diagnosed as euploid by aCGH were reanalyzed using NGS. Cytogenetic reports of the products of conception for 20 of the pregnancies resulting in miscarriage were available for comparison. MAIN OUTCOME MEASURE(S) The incidence of aneuploidy and mosaicism using NGS within embryos resulting in miscarriage and live birth. RESULT(S) Of euploid embryos analyzed by aCGH resulting in miscarriage, 31.6% were mosaic and 5.2% were polyploid by NGS. The rate of chromosomal abnormalities was significantly higher in embryos resulting in miscarriage (36.8%) than in those resulting in live births (15.8%). The rate of mosaicism was twice as high among embryos resulting in miscarriage than those resulting in live birth, but this was not statistically significant. Next-generation sequencing detected more cases of mosaicism than cytogenetic analysis of products of conception. CONCLUSION(S) Undetected aneuploidy may increase the risk of first trimester pregnancy loss. Next-generation sequencing may detect mosaicism and triploidy more frequently than aCGH, which could help to identify embryos at high risk of miscarriage. Mosaic embryos, however, should not be discarded as some can result in live births.

Sperm-induced currents at fertilization in sea urchin eggs injected with EGTA and neomycin

Membrane currents were measured in single voltage-clamped sea urchin eggs (Lytechinus pictus and Lytechinus variegatus) that were injected with either EGTA or neomycin and inseminated. Although egg activation and the fertilization calcium wave were prevented by injection of either of these compounds, sperm attached and still elicited inward currents. Sperm-induced currents in EGTA-injected eggs had an abrupt onset, quickly reached a maximum, and then slowly declined in amplitude. Sperm incorporation occurred readily in EGTA-injected eggs. Similar results were obtained with another calcium chelator, BAPTA. In neomycin-injected eggs, sperm-induced currents generally had an abrupt onset and, in contrast to EGTA-injected eggs, the currents usually cut off rapidly. Sperm failed to enter the neomycin-injected eggs and the duration of sperm-induced currents in neomycin-injected eggs was markedly dependent upon the voltage-clamp holding potential, with shorter duration currents occurring at -70 than at -20 mV. The lability of the initial interaction between sperm and egg at negative holding potentials may explain why activation often fails when the egg membrane is voltage clamped at these potentials (Lynn et al., Dev. Biol. 128, 305-323, 1988).

Assessing morphokinetic parameters via time lapse microscopy (TLM) to predict euploidy: are aneuploidy risk classification models universal?

PurposeTo determine if Aneuploidy Risk Classification Models are predictive of euploidy/aneuploidy amongst IVF facilities.MethodsWe retrospectively applied key time lapse imaging events of embryos (Campbell et al.[5, 6]) to stratify embryos into 3 groups: low, medium and high risk of aneuploidy. The actual ploidy results (from array comparative genomic hybridization) were compared with expectations [5, 6]. Sources of variability in morphokinetic parameters were determined using Analysis of Variance (ANOVA).ResultsThe model failed to segregate euploid embryos from aneuploid embryos cultured at our facility. Further analysis indicated that the variability of embryos among patients was too great to allow selection of euploid embryos based on simple morphokinetic thresholds. Clinical selection of embryos based on morphokinetics alone is unlikely to identify euploid embryos accurately for transfer or yield higher rates of live delivery.ConclusionsThe use of non-invasive morphokinetics is unlikely to discriminate aneuploid from euploid embryos. Further, it does not approach the accuracy of preimplantation genetic screening with array comparative genomic hybridization.

Membrane depolarization facilitates sperm entry, large fertilization cone formation, and prolonged current responses in sea urchin oocytes

Depolarization of the sea urchin eggs membrane is required for two processes during fertilization: the entry of the fertilizing sperm and the block to polyspermy which prevents the entry of supernumerary sperm. In an immature sea urchin oocyte, the depolarization is very small in response to the attachment of a sperm. The purpose of this study was to determine whether the depolarization evoked by sperm attaching to an oocyte can facilitate sperm entry or induce the block to polyspermy. Individual oocytes of the sea urchin with diameters which ranged from 86 to 102% that of the average diameter for mature eggs from the same female were examined. The oocytes have a membrane potential of -73 +/- 6 mV (SD, n = 80) and a very low input resistance compared to that of mature eggs. Single sperm, following attachment to an oocyte, elicit a brief, small depolarization with a maximum amplitude of 8 +/- 1.4 mV (SE, n = 15), frequently followed by the formation of tiny filament-like fertilization cones, but the sperm fail to enter. If oocytes are voltage-clamped at membrane potentials more negative than -20 mV, following attachment of the sperm small transient inward currents occur, similar filament-like cones form, and the sperm do not enter. When many sperm attach to an oocyte which is not voltage clamped, the depolarizations sum to create a large depolarization with an amplitude of 60 to 80 mV, which shifts the oocytes membrane potential to a value between -10 and +5 mV; more positive values are not attained. At such membrane potentials, whether the potential is maintained by the summed depolarizations of many attached sperm or by voltage clamp, large fertilization cones form, the sperm enter, and the oocytes can become highly polyspermic. In oocytes voltage clamped at +20 mV, however, both sperm entry and fertilization cone formation are suppressed. Therefore, both types of voltage-dependence for sperm entry are present in oocytes, although the depolarization caused by a single sperm is not large enough to permit its entry, nor is the depolarization caused by many sperm sufficient to prevent the entry of supernumerary sperm.

Progesterone receptor membrane component-1 regulates hepcidin biosynthesis

Iron homeostasis is tightly regulated by the membrane iron exporter ferroportin and its regulatory peptide hormone hepcidin. The hepcidin/ferroportin axis is considered a promising therapeutic target for the treatment of diseases of iron overload or deficiency. Here, we conducted a chemical screen in zebrafish to identify small molecules that decrease ferroportin protein levels. The chemical screen led to the identification of 3 steroid molecules, epitiostanol, progesterone, and mifepristone, which decrease ferroportin levels by increasing the biosynthesis of hepcidin. These hepcidin-inducing steroids (HISs) did not activate known hepcidin-inducing pathways, including the BMP and JAK/STAT3 pathways. Progesterone receptor membrane component-1 (PGRMC1) was required for HIS-dependent increases in hepcidin biosynthesis, as PGRMC1 depletion in cultured hepatoma cells and zebrafish blocked the ability of HISs to increase hepcidin mRNA levels. Neutralizing antibodies directed against PGRMC1 attenuated the ability of HISs to induce hepcidin gene expression. Inhibiting the kinases of the SRC family, which are downstream of PGRMC1, blocked the ability of HISs to increase hepcidin mRNA levels. Furthermore, HIS treatment increased hepcidin biosynthesis in mice and humans. Together, these data indicate that PGRMC1 regulates hepcidin gene expression through an evolutionarily conserved mechanism. These studies have identified drug candidates and potential therapeutic targets for the treatment of diseases of abnormal iron metabolism.

Sperm Entry in Sea Urchin Eggs: Recent Inferences Concerning its Mechanism

The mechanism by which a sperm enters the cytoplasm of an egg during fertilization is not known; but, inn the sea urchin egg, membrane potentials in two different ranges are capable of precluding sperm entry. we have attempted to determine what steps of sperm entry are regulated by membrane potential in eggs and oocytes of Lytechinus variegatus with the objective of obtaining insight into the sperm entry process. Early sperm-egg interactions include: sperm-egg attachment or binding, sperm-egg fusion, and an abrupt increase of membrane conductance which leds to a depolarization of the egg’s membrane potential. The positive-going activation potentail of the sea urchun egg following attachment of the first sperm decreases the probability of entry for sperm which attach subsequently (rapid, voltage block to polyspermy). A sperm fails to enter the egg or oocyte unless it first causes a conductance increase. In voltage-clamped eggs and oocytes, the probability that an attached sperm causes a conductance increase is maximal at potentials more negati ve than -10m V and decreases to 0 near +20 m V. It is possible that the lack of a conductance increase at potentials more positive than +20 m V results from a voltage-induced failure of sperm to fuse with the egg following attachment, hence precluding their entry. When sperm entry occurs at less positive potentials, the sperm’s nucleus slowly migrates into the cytoplasm of the egg beginning roughly 60s after the initial conductance increase. However, if an egg or oocyte is voltage clamped at a potential more negative than -30 mV (their resting potentials are -75 m V), it is unlikely that a sperm which attaches and causes a conductance increase will be incorporated. A characteristic “cutoff” of the conductance increase occurs in association with the failure of these sperm to enter. In oocytes which are not voltage clamped, depolarizations associated with attachment of a single sperm also cut off prematurely and are too small topermit sperm incorporation. Sperm entry occurs in oocytes only when many sperm attach and involves a cooperative summation of their depolarizations. Cooperativity is not seen for eggs. In oocytes, sperm incorporation, fertilization cone size, and the duration of the electrophysiological response are statistically associated and exhibit voltage dependences which are indistinguishable, suggesting that the three events are interrelated. We conclude that sperm entry involves two voltage-dependent, separable steps: sperm-egg fusion and nuclear incorporation. One common, voltage dependent mechanism may regulate nuclear incorporation and fertilization cone formation.