The impact of male factor infertility on early and late morphokinetic parameters: a retrospective analysis of 4126 time-lapse monitored embryos.

STUDY QUESTION: Is there an effect of male factor infertility (MFI) on either early or late morphokinetic parameters obtained during embryonic culture to blastocyst stage in a time-lapse imaging (TLI) incubator? SUMMARY ANSWER: Neither mild nor severe MFI had an impact on overall time to blastocyst or duration of individual cleavage stages in the total embryo population. WHAT IS KNOWN ALREADY: Prior studies have suggested that paternal DNA and sperm quality affect embryo morphokinetic parameters, but the impact of MFI is not fully understood. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study, at a major academic fertility centre, included 536 couples (women, ≤44 years of age) undergoing IVF between September 2013 and September 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data from 4126 embryos cultured to the blastocyst stage in a TLI-monitored incubator were retrospectively reviewed. Embryos derived from the sperm of men with MFI were compared with those derived from patients with other infertility diagnoses. Generalized fixed and random effects models, t-test and χ2 were used as appropriate. MAIN RESULTS AND THE ROLE OF CHANCE: Couples with MFI had a higher rate of ICSI utilization and fewer usable embryos on average, and the men were older compared with couples with other diagnoses. Additionally, the women in MFI couples were younger and had higher antral follicle counts (AFCs) and higher anti-Müllerian hormone (AMH) levels compared with the other women undergoing IVF. When controlling for maternal and paternal ages, AMH and fertilization method (conventional IVF versus ICSI), neither mild nor severe MFI affected duration of individual cleavage stages or overall time to the blastocyst stage, when all or only usable embryos were examined (coefficient 0.44 hours in all embryos, P = 0.57; coefficient 0.39 hours in usable embryos, P = 0.60). Whether the sperm was surgically extracted similarly had no significant effect on embryo morphokinetic parameters. When the fertilization method was assessed independently, ICSI lengthened the overall time to blastocyst stage by 1.66 hours (P = 0.03) on average, primarily due to an increase in duration of the time from 5-cell embryo stage to early blastulation (P5SB). LIMITATIONS, REASONS FOR CAUTION: This large cohort study avoided embryo selection bias due to random assignment of embryos to the TLI incubators. However, our findings may not be generalizable to groups under-represented in our clinic population. Future studies should also evaluate the impact of male hormonal status and detailed sperm morphology, such as head versus flagellum defects, on embryo morphokinetic development. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest that the fertilization method rather than MFI per se impacts time to early blastulation. The clinical implications of this effect on embryo development warrant further investigation. STUDY FUNDING/COMPETING INTEREST(S): There were no sources of funding for this study. There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Measurement of steady-state growth rates of a thermophilic alga directly in nature.

Steady-state growth rates of thermophilic algae can be determined directly in nature in the flowing waters of a hot spring channel by measuring the rate of loss of algal cells when the channel is darkened. The half time of the loss rate in the steady state is identical to the generation time. We studied the unicellular blue-green alga Synechococcus in Yellowstone National Park. Temperature and flow rate remained relatively constant throughout the experiment. Quantitative cores were taken and homogenized; the algal cells were then counted by use of a Petroff-Hausser counting chamber. After ascertaining that the algal population was in a steady state, the channel was darkened in such a way that neither flow rate nor temperature was altered. The population began to decrease within 1 day; the loss rate was exponential with time for 2 to 3 decades. Half times were then calculated from these loss rates. The growth rates found were considerably lower than those for the same organism in laboratory culture. The results suggest that in nature the organism may be an obligate phototroph. In two cases, after the algal populations decreased to an undetectable level, the dark covers were removed and the rate of recolonization was measured. The kinetics of recolonization were different from the kinetics of washout.

Estrogen stabilizes vitellogenin mRNA against cytoplasmic degradation.

We have demonstrated, by DNA excess filter hybridizations to pulse-labeled cell RNA, that estrogen selectively stabilizes Xenopus liver vitellogenin mRNA against cytoplasmic degradation. The half-life of vitellogenin mRNA is approximately 3 weeks in the presence of estrogen and 16 hr after estrogen is withdrawn from the culture medium. Total poly(A) mRNA exhibits the same half-life (16 hr) in the presence or absence of estrogen. The rapid cytoplasmic degradation of vitellogenin mRNA in the absence of estrogen is fully reversible upon restimulation with estrogen, indicating that nuclear modification of vitellogenin RNA transcripts is not responsible for their stability. Intermediate levels of vitellogenin mRNA stability and changes in the relative rate of vitellogenin gene transcription are not observed late in estrogen induction, when vitellogenin mRNA levels plateau. Instead, Xenopus liver cells achieve fine control over the level of vitellogenin mRNA through down-regulation of the overall rate of total nuclear RNA synthesis.

Estrogen regulates the absolute rate of transcription of the Xenopus laevis vitellogenin genes.

Estrogen regulates the synthesis of the egg yolk precursor protein, vitellogenin, by causing both a 20-60-fold increase in the absolute rate of total nuclear RNA synthesis and a selective increase of at least several thousand fold in the absolute rate of vitellogenin gene transcription. Vitellogenin gene transcription is undetectable in unstimulated and withdrawn Xenopus laevis liver cells and in cultured Xenopus kidney cells allowing us to set a very low upper limit (less than 1 transcript/vitellogenin gene/day) on potential basal rates of vitellogenin gene transcription. The elevated rates of vitellogenin mRNA accumulation previously observed during restimulation of withdrawn liver cells (secondary estrogen stimulation) appear to be due to an increased rate of vitellogenin gene transcription. Both the maximum transcription rate and the rapidity of the early response increase on secondary estrogen stimulation. Relative transcription rates were determined by hybridization of pulse-labeled nuclear RNA to vitellogenin cDNA clones immobilized on nitrocellulose filters. The conversion of relative transcription rates to absolute transcription rates, was facilitated by development of a sensitive high performance liquid chromatography method for quantitation of the specific radioactivity of the cellular UTP pool.

RECOVERY OF A HOT SPRING COMMUNITY FROM A CATASTROPHE.

The algal mats of a number of hot springs in the Lower Geyser Basin of Yellowstone National Park were destroyed by a brief violent hailstorm on August 30, 1967. The rate of recovery of the algal mat at Mushroom Spring was studied by quantitative methods. In the temperature range of 65-71 C a unicellular cyanophycean alga is the sole photosynthetic component. The doubling times during the recovery period for three stations were: Station I (71 C), 17 days; station II (68 C), 10.5 days; station III (65 C), 10 days. The algal mat had returned to apparently normal size by 152 days after the catastrophe. The significance of these observations for the conservation of hot spring communities is discussed.

Activation of vitellogenin gene transcription is a direct response to estrogen in Xenopus laevis liver.

Estrogen induces the synthesis of vitellogenin mRNA by activating transcription of the vitellogenin genes. Quantitative inhibition of liver protein synthesis by cycloheximide does not prevent activation of vitellogenin gene transcription. The relative transcription rate of the vitellogenin genes in estrogen stimulated liver is similar in control and cycloheximide treated animals (800-1000 ppm). Selective estrogen activation of vitellogenin gene transcription therefore represents a direct effect of estrogen on vitellogenin gene transcription which can occur without any change in the cells' protein complement. Two other cellular responses to estrogen, the induction of nuclear estrogen receptor, and an increased rate of total nuclear RNA synthesis, are blocked by cycloheximide administration. Since the overall rate of vitellogenin mRNA synthesis is a function of both the selective estrogen activation of vitellogenin gene transcription which is not blocked by cycloheximide and the increased rate of total nuclear RNA synthesis which is blocked by cycloheximide, the total rate of vitellogenin mRNA synthesis is markedly reduced following cycloheximide administration.

The role of estrogen receptor in Xenopus laevis vitellogenin gene expression.

Administration of estradiol 17 beta [estra-1,3,5(10)-triene-3,17-beta-diol] to male Xenopus laevis induces the massive synthesis by the liver of the egg yolk precursor phospholipoglycoprotein, vitellogenin, and its cognate mRNAs. Restimulation of male X. laevis that have been previously induced to synthesize vitellogenin mRNA but are inactive in vitellogenin mRNA synthesis at the time of restimulation with estrogen results in more rapid accumulation of vitellogenin mRNA and more efficient transcription of the vitellogenin genes than occurs following primary estrogen stimulation. The estrogen receptor system that mediates estrogen action in this organism exhibits several unusual properties. The cytoplasm of unstimulated liver cells contains high levels of a middle-affinity estrogen-specific binding protein and little if any estrogen receptor. The properties of the estrogen binding protein are consistent with a role in protecting estradiol 17 beta against metabolism, as a fraction of cytoplasmic estradiol 17 beta is not subject to rapid metabolism. In addition, similar binding activities are found in all Xenopus tissues surveyed that respond to steroid hormones. The induction of nuclear estrogen receptor is coincident with the onset of vitellogenin mRNA accumulation. However, an increased level of estrogen receptor is not responsible for the elevated rate of vitellogenin gene transcription observed following restimulation with estrogen.

Giant heterogeneous polyadenylic acid on vesicular stomatitis virus mRNA synthesized in vitro in the presence of S-adenosylhomocysteine.

An in vitro transcription system in which vesicular stomatitis virus (VSV) mRNA species have been synthesized is described. In addition to purified VSV virions, which contain an RNA-dependent RNA polymerase, this system contained a cytoplasmic cell extract that enhanced correct transcription. Gel electrophoretic analysis of the methylated polyadenylic acid [poly(A)]-containing VSV mRNA produced in this system in the presenct of S-adenosylmethionine showed the discrete VSV mRNA species. However, when unmethylated mRNA was synthesized in the presence of S-adenosylhomocysteine, the poly(A)-containing transcripts were large and heterogeneous in molecular weight and did not contain discrete VSV mRNA species. Two-dimensional fingerprint analysis of the methylated and unmethylated products suggested that identical nucleotide sequences were present in the RNAs. Further analysis showed the presence of very large heterogeneous poly(A), 200 to 2,000 nucleotides in lenght, in the unmethylated transcript. Proof that this large poly(A) was covalently linked to the correct VSV mRNA transcripts was obtained by removal of the poly(A) by hybirdization with oligodeoxythymidylic acid and digestion with RNase H. This digestion produced unmethylated VSV mRNA transcripts with the same discrete sizes as the deadenylated RNAs produced from VSV mRNA initially isolated from VSV-infected cells. The results suggest that there is a relationship between methylation at the 5'-end and polyadenylation at the 3'-end of VSV mRNA's. Furthermore, addition of the very large poly(A) does not affect the normal process of sequential transcription of the VSV genome, suggesting that this poly(A) addition is occurring independently of further transcription.

Microbial life at 90 C: the sulfur bacteria of Boulder Spring.

The physiology of the bacteria living in Boulder Spring (Yellowstone National Park) at 90 to 93 C was studied with radioactive isotope techniques under conditions approximating natural ones. Cover slips were immersed in the spring; after a fairly even, dense coating of bacteria had developed, these cover slips were incubated with radioactive isotopes under various conditions and then counted in a gas flow or liquid scintillation counter. Uptake of labeled compounds was virtually completely inhibited by formaldehyde, hydrochloric acid, and mercuric bichloride, and inhibition was also found with streptomycin and sodium azide. The water of Boulder Spring contains about 3 mug of sulfide per ml. Uptake of labeled compounds occurs only if sulfide or another reduced sulfur compound is present during incubation. The pH optimum for uptake of radioactive compounds by Boulder Spring bacteria is 9.2, a value near that of the natural spring water (8.9). Many experiments with a variety of compounds were performed to determine the temperature optimum for uptake of labeled compounds. The results with all the compounds were generally similar, with broad temperature optima between 80 and 90 C, and with significant uptake in boiling (93 C) but not in superheated water (97 C). The results show that the bacteria of Boulder Spring are able to function at the temperature of their environment, although they function better at temperatures somewhat lower. The fine structure of these bacteria has been studied by allowing bacteria in the spring to colonize glass slides or Mylar strips which were immediately fixed, and the bacteria were then embedded and sectioned. The cell envelope structure of these bacteria is quite different from that of other mesophilic or thermophilic bacteria. There is a very distinct plasma membrane, but no morphologically distinct peptidoglycan layer was seen outside of the plasma membrane. Instead, a rather thick diffuse layer was seen, within which a subunit structure was often distinctly visible, and connections frequently occurred between this outer layer and the plasma membrane. The thick outer layer usually consisted of two parts, the outer part of which was sometimes missing. Within the cells, structures resembling ribosomes were seen, and regions lacking electron density which probably contained deoxyribonucleic acid were also visible.