Effect of ejaculatory abstinence period on fertilization and clinical outcomes in ICSI cycles: a retrospective analysis.

RESEARCH QUESTION: Does ejaculatory abstinence impact fertilization outcomes in intracytoplasmic sperm injection (ICSI) cycles in infertile couples? DESIGN: This single-centre retrospective observational study included 6919 ICSI cycles from 2013 to 2022. The primary outcome was the assessment of oocyte fertilization, measured in terms of the rate of formation of two-pronuclear (2PN), 3PN and 1PN zygotes. Secondary outcomes were blastulation, cumulative positive ß-human chorionic gonadotrophin test and clinical pregnancy rates. Relationships between ejaculatory abstinence and fertilization outcomes, and ejaculatory abstinence and clinical outcomes were evaluated with multivariable analysis, including possible confounders. RESULTS: A positive association was observed between ejaculatory abstinence and semen sample volume (P < 0.001), sperm concentration (P < 0.001) and total motile sperm count (P < 0.001). No association was found between the 1PN zygote rate and ejaculatory abstinence (P = 0.97). Conversely, for each additional day of ejaculatory abstinence, the likelihood of obtaining 2PN zygotes from all inseminated oocytes decreased by 3% [adjusted odds ratio (aOR) 0.97, 95% CI 0.94-0.99], whilst the likelihood of obtaining 3PN zygotes from all inseminated oocytes increased significantly by 14% (aOR 1.14, 95% CI 1.07-1.22). No significant associations were found between ejaculatory abstinence and blastulation, cumulative pregnancy or miscarriage rates. CONCLUSIONS: A longer ejaculatory abstinence period significantly decreases the rate of 2PN zygotes, and increases the rate of 3PN zygotes without directly affect blastulation and pregnancy rates.

Multifactorial analysis of the stochastic epigenetic variability in cord blood confirmed an impact of common behavioral and environmental factors but not of in vitro conception.

Background: An increased incidence of imprint-associated disorders has been reported in babies born from assisted reproductive technology (ART). However, previous studies supporting an association between ART and an altered DNA methylation status of the conceived babies have been often conducted on a limited number of methylation sites and without correction for critical potential confounders. Moreover, all the previous studies focused on the identification of methylation changes shared among subjects while an evaluation of stochastic differences has never been conducted. This study aims to evaluate the effect of ART and other common behavioral or environmental factors associated with pregnancy on stochastic epigenetic variability using a multivariate approach. Results: DNA methylation levels of cord blood from 23 in vitro and 41 naturally conceived children were analyzed using the Infinium HumanMethylation450 BeadChips. After multiple testing correction, no statistically significant difference emerged in the number of cord blood stochastic epigenetic variations or in the methylation levels between in vitro- and in vivo-conceived babies. Conversely, four multiple factor analysis dimensions summarizing common phenotypic, behavioral, or environmental factors (cord blood cell composition, pre or post conception supplementation of folates, birth percentiles, gestational age, cesarean section, pre-gestational mother's weight, parents' BMI and obesity status, presence of adverse pregnancy outcomes, mother's smoking status, and season of birth) were significantly associated with stochastic epigenetic variability. The stochastic epigenetic variation analysis allowed the identification of a rare imprinting defect in the locus GNAS in one of the babies belonging to the control population, which would not have emerged using a classical case-control association analysis. Conclusions: We confirmed the effect of several common behavioral or environmental factors on the epigenome of newborns and described for the first time an epigenetic effect related to season of birth. Children born after ART did not appear to have an increased risk of genome-wide changes in DNA methylation either at specific loci or randomly scattered throughout the genome. The inability to identify differences between cases and controls suggests that the number of stochastic epigenetic variations potentially induced by ART was not greater than that naturally produced in response to maternal behavior or other common environmental factors.

Principles of phenomics in endometriosis.

BACKGROUND: Endometriosis has been associated with specific morphometric characteristics and pigmentary traits. The purpose of this study was to systematically review prior publications dealing with this aspect in order to revisit phenotypic information in the context of phenomics principles. METHODS: Comprehensive searches of Pubmed, Medline and Embase were conducted to identify studies, published from 1990 to 2011 in the English language literature, on the relationship between endometriosis and morphometric characteristics/pigmentary traits. RESULTS: We identified 11 studies on the association between endometriosis and body mass index (BMI) in the adult population and 5 studies on the same association during early life. While a modest inverse correlation was found between endometriosis and adult BMI, a stronger association was consistently demonstrated between endometriosis and early life body size, even after adjusting for confounding factors such as age, birthweight, age at menarche, parity and oral contraceptive use. Four papers have been published on the association between endometriosis and cutaneous naevi and five on the association between the disease and specific pigmentary traits. A skin phenotype characterized by the presence of naevi and freckles and by a high sensitivity to sun exposure is represented more frequently in women with endometriosis. CONCLUSIONS: Endometriosis appears to be associated with some phenotypic variations likely attributable to the strong effect of the environment on the expression and function of genes influencing the traits. Novel clues on endometriosis pathogenesis may derive from the analysis of the phenotypic traits associated with the disease.

Epigenetic analysis of the critical region I for premature ovarian failure: demonstration of a highly heterochromatic domain on the long arm of the mammalian X chromosome.

BACKGROUND: X chromosome rearrangements defined a critical region for premature ovarian failure (POF) that extended for >15 Mb in Xq. It has been shown previously that the region could be divided into two functionally distinct portions and suggested that balanced translocations interrupting its proximal part, critical region 1 (CR1), could be responsible for POF through downregulation of ovary expressed autosomal genes translocated to the X chromosome. RESULTS AND CONCLUSION: This study reports that such position effect can indeed be demonstrated by analysis of breakpoint regions in somatic cells of POF patients and by the finding that CR1 has a highly heterochromatic organisation, very different from that of the euchromatic autosomal regions involved in the rearrangements. The chromatin organisation of the POF CR1 is likely to be responsible for the epigenetic modifications observed in POF patients. The characteristics of CR1 and its downregulation in oocytes may very well explain its role in POF and the frequency of the POF phenotype in chromosomal rearrangements involving Xq. This study also demonstrates a large and evolutionary conserved domain of the long arm of the X chromosome, largely corresponding to CR1, that may have structural or functional roles, in oocyte maturation or in X chromosome inactivation.

Objective evaluation of the viability of cryopreserved oocytes.

Recent studies of fundamental cryobiology, empirical observations and more systematic clinical experiences have generated a renewed interest in oocyte cryopreservation. Poor survival rate has long been the limiting factor which has prevented widespread adoption of oocyte storage. Slow-cooling and vitrification protocols developed in the last few years have apparently solved this problem, ensuring high recovery of viable oocytes from liquid nitrogen storage. However, the definition of oocyte viability appears rather vague. In fact, post-storage survival as assessed on morphological criteria, indicated by the absence of overt cell degeneration, is not necessarily synonymous with viability. Despite its sensitivity to low temperatures, the meiotic spindle can be preserved after cryopreservation and its constitution after thawing can be monitored non-invasively through polarized light microscopy. Assessment of oocyte cryopreservation via clinical parameters is a daunting task. Most studies are small and difficult to interpret because of confounding factors, such as age, patient selection and quality and strategy of use of the cryopreserved material. Some progress has been made, however, as suggested by recent experiences in which the implantation efficiency of embryos produced from thawed oocytes approaches that reported using cryopreserved embryos directly.

Oocyte cryopreservation: clinical outcome of slow-cooling protocols differing in sucrose concentration.

Oocyte cryopreservation represents an important option for management of female fertility, avoiding the ethical concerns associated with embryo storage. This retrospective study evaluated the clinical outcome of two alternative slow freezing protocols involving different sucrose concentrations. From January 2004 to March 2006, spare oocytes from selected couples undergoing IVF or intracytoplasmic sperm injection were frozen using a slow-cooling protocol and thawed at a later stage. Patients were divided into two groups: group A (n = 65), whose oocytes were frozen with propane-1,2-diol (PrOH) and 0.1 mol/l sucrose; and group B (n = 66) whose oocytes were frozen with 0.3 mol/l sucrose. A total of 543 oocytes were thawed in group A and 601 in group B, achieving a survival rate of 24.3 and 71.2% respectively. Whilst fertilization rate (53.5 and 80.4% respectively) was higher in group B, enhanced results for group A were achieved over all (implantation rate per transferred embryos 12.2 versus 5.7%; pregnancy rate per transfer 16.7 versus 9.5%). Normal births and ongoing pregnancies have occurred in both groups. Although in slow-cooling methods higher sucrose concentration in the freezing mixture allows higher post-thaw survival and fertilization rates, overall this did not coincide with an improved clinical outcome.

Serum progesterone at human chorionic gonadotropin injection significantly correlate with female age.

AIM: In in vitro fertilization-embryo transfer (IVF-ET) higher age and low responses are associated with accelerated luteinization of mature follicles rather than diminished responsiveness. The aim of this study was to determine whether an elevated serum progesterone (P) on the day of human chorionic gonadotropin (hCG) administration during gonadotropin stimulation for IVF-ET is associated with age. METHODS: E2 (17beta estradiol) and P concentrations on the day of hCG administration, number and quality of oocytes and embryos, and clinical pregnancies were retrospectively analyzed in 460 women undergoing IVF-ET. We evaluated patients according to age; the 25-30 age group (n=140), the 31-35 age group (n=100), the 36-40 (n=90), and the 41-45 age group (n=130). RESULTS: In the 25-30 age group (n=140) P was 0.67+/-0.3 ng/mL, in the 31-35 age group (n=100) P was 0.87+/-0.2 ng/mL, in the 36-40 age group (n=90) P was 0.95+/-0.2 ng/mL, in the 41-45 age group (n=130) P was 1+/-0.2 ng/mL. The difference between the 25-30 age group and the 41-45 age group was statistically significant (P<0.05). CONCLUSIONS: Periovulatory levels of serum P vary according to ovarian response to controlled ovarian hyperstimulation. Periovulatory P may reflect inadequate steroidogenesis. In women stimulated with recombinant follicle stimulating hormone for IVF, the serum P on the day of hCG administration increases with age.

Polyamine oxidase and tissue transglutaminase activation in rat small intestine by polyamines.

Polyamine degradation was studied in the small intestine from rats fed on a polyamine-supplemented diet. Lactalbumin diet was given to Hooded-Lister rats, with or without 5 mg rat(-1) day(-1) of putrescine or spermidine for 5 days. Polyamine oxidase activity increased with putrescine and spermidine in the diet, whereas spermidine/spermine N(1)-acetyltransferase and diamine oxidase activities were unchanged. We also studied the calcium-dependent and -independent tissue transglutaminase activities, since they can modulate intestinal polyamine levels. Both types of enzymes increased in the cytosolic fraction after putrescine (about 65%) or spermidine (80-100%). Our results indicate that exogenous polyamines stimulate intestinal polyamine oxidase and tissue transglutaminase activities, probably to prevent polyamine accumulation, when other pathways of polyamine catabolism (acetylation and terminal catabolism) are not activated.

Distribution and activity of transglutaminase in rat brain carcinogenesis and in gliomas.

Tissue transglutaminase is a calcium-dependent enzyme which may influence cell morphology, cytoskeletal processes and membrane functions. During rat brain carcinogenesis induced by transplacental administration of N-ethyl-N-nitrosourea to BD IX rats, cytosolic tissue transglutaminase activity was increased by about 140% at 30 days of extrauterine life and returned towards the control values at 3-5 months. In the particulate fraction, enzyme activity progressively increased, reaching values similar to those present in the developed gliomas. Tissue transglutaminase activity in gliomas had a behavior inverse to that observed in controls, with a decrease (about 50%) in the cytosol and a marked increase (380%) in the particulate fraction, indicating a redistribution of enzyme activity.

In vivo modulation of 73 kDa heat shock cognate and 78 kDa glucose-regulating protein gene expression in rat liver and brain by ethanol.

BACKGROUND: In cultured cells of various origin, ethanol induces the synthesis of 70 kDa family heat shock proteins (hsp70 family), which play a role in the protection of protein traffic and secretion, as well as in cytoskeleton organization. To assess whether ethanol also can induce such genes in vivo, we studied the behavior of hsp70, hsc73, and grp78 messenger ribonucleic acids (mRNAs) and related proteins in the liver and brain of rats acutely treated with ethanol. METHODS: Overnight fasted Sprague-Dawley rats (220-250 g) were acutely treated with a low (2 g/kg body weight) or a high (5 g/kg body weight) dose of ethanol as a 30% solution in saline or an equal volume of saline (controls) by gastric intubation. Animals were killed at various times after treatments (3-72 hr). Messenger RNA levels for different members of hsp70 family (hsp70; 73 kDa heat shock cognate, or hsc73; and 78 kDa glucose-regulating protein, or grp78) were determined by Northern blot analysis and hybridization with specific complementary deoxyribonucleic acid (cDNA) probes. The amounts of related proteins were assayed by Western blot analysis with specific antibodies. Autoradiograms and fluorograms were subjected to densitometric scanning. RESULTS: Ethanol (2 g/kg) caused a slight increase in hsc73 and grp78 mRNA levels only in the liver, without enhancing the amount of proteins. Ethanol (5 g/kg) increased the level of hsc73 and grp78 mRNAs and related proteins in the liver. In the brain, the amount of hsc73 mRNA was enhanced, but this did not change hsc73 protein. In addition, we observed an increase in cerebral grp78 transcript and related protein. Hsp70 gene was not induced in the examined tissues by either dose of ethanol. CONCLUSIONS: Hepatic and cerebral hsc73 and grp78 genes are responsive to ethanol in vivo, and their activation may signal the cell's effort to counteract the harmful action of ethanol.

Changes in hepatic polyamine catabolism in elderly rats.

AIMS/BACKGROUND: Given the important role of polyamines (putrescine, spermidine, spermine) in the modulation of macromolecular syntheses, gene expression and proteolysis, alterations in their metabolic pathways could be relevant during senescence. Since the few existing data address mainly polyamine biosynthesis, we studied the oxidative catabolism of polyamines in the liver of rats 3-36 months of age. METHODS: Polyamine oxidase activity was fluorimetrically measured using N1-acetylspermine as substrate. Spermidine/spermine N1-acetyltransferase and diamine oxidase were measured by radiochemical methods using labeled acetyl-coenzyme A and putrescine, respectively, as substrate. Polyamines were separated by HPLC and fluorimetrically quantified after post-column derivatization with o-phthaldialdehyde. RESULTS: Spermidine/spermine N1-acetyltransferase activity increased in 36-month-old rats and polyamine oxidase activity in 24- and 36-month-old rats. A decline in spermine and increases in spermidine and putrescine in elderly rats suggested an activation of the interconversion pathway of higher into lower polyamines. The activity of diamine oxidase, which degrades putrescine, was enhanced starting from 12 months of age. CONCLUSION: In the liver of aged rats, an increase in the catabolic enzymes leads to a reconversion of the higher polyamines to putrescine. This increased catabolism may represent an important age-related change and may contribute to impairment of the expression of growth-related genes in senescence.

Oxidative degradation of polyamines in rat pancreatic hypertrophy.

In the hypertrophic pancreas, we studied the oxidative degradation of polyamines, which are endogenous polycations important for cell division, growth and differentiation. To induce pancreatic hypertrophy, rats were fed on a semi-synthetic diet containing a daily dose of 42 mg phytohaemagglutinin per rat for 5 or 10 days. In the model, the activities of polyamine oxidase (the enzyme that degrades spermidine, spermine and mainly their acetyl derivatives) and diamine oxidase (the key enzyme of terminal catabolism of polyamines in vivo) increased by 100-180% and 90-100%, respectively, parallel to an elevation in polyamine content (40-100%). The results suggest that in pancreas hypertrophy, which does not exhibit stimulation of spermidine/spermine N1-acetyltransferase activity, increases in the activity of polyamine and diamine oxidases are related events that lead to putrescine formation and removal of excess polyamines.

Transglutaminase activity in rat brain after ethanol exposure.

The effect of acute and chronic ethanol treatment on the activity of tissue transglutaminase (a calcium-dependent enzyme that catalyzes the covalent association between proteins, as well as proteins and polyamines) was studied in homogenate and in the cytosolic fraction of rat brain (telencephalon and diencephalon). A single dose of ethanol (5 g/kg of body weight, by gastric intubation) caused a 2-fold increase in enzyme activity at 6 hr after the ethanol dose, with a return toward the basal values at 24 hr. In vitro experiments with ethanol or acetaldehyde showed that the increase in transglutaminase activity was due to ethanol per se and not to its metabolism. The enzyme stimulation was correlated with a decrease in the levels of the polyamine spermine, a physiological substrate for the enzyme. Similar results were also found in the brain from rats fed on an ethanol diet for 4 months. The enhancement in tissue transglutaminase activity may thus lead to a decline in spermine, a polyamine known to have important protective functions in the cell.

Response of intestinal transglutaminase activity to dietary phytohaemagglutinin.

The behaviour of the activity of tissue transglutaminase, a calcium-dependent enzyme, and the levels of polyamines which are physiological substrates for the enzyme, were studied in rat small intestine induced to grow by lectin phytohaemagglutinin. Transglutaminase activity greatly increased in the homogenates and the cytosolic fractions of the intestinal mucosa of lectin-treated rats compared to that of untreated animals. The measurement of enzyme activity in the presence of monodansylcadaverine, a competitive inhibitor of transglutaminase, testified that the assayed enzyme activity was authentic transglutaminase. As regards polyamines, the level of spermine did not change, whereas putrescine and spermidine contents were enhanced. The activation of transglutaminase, which was probably due to Ca2+ accumulation in enterocytes, could have a role in maintaining enterocyte adhesion and intestinal cell homeostasis, and/or repairing lectin-induced damages of microvilli of the gut epithelium.