Profiling of mitochondrial heteroplasmy in single human oocytes by next-generation sequencing.

Mitochondrial DNA (mtDNA) plays a crucial role in the development of a competent oocyte. Indeed, mtDNA alterations may predispose to chromosome nondisjunction, resulting in infertility due to a reduced vitality and quality of oocytes and embryos. In this methods paper, the multiple displacement amplification approach was applied in combination with next-generation sequencing (NGS) to amplify and sequence, in single-end, the entire mtDNA of single human oocytes to directly construct genomic NGS libraries, and subsequently, to highlight and quantify the mutations they presented. The bioinformatic workflow was carried out with a specific ad hoc developed in-house software. This approach proved to be sensitive and specific, also highlighting the mutations present in heteroplasmy, showing deletion, insertion or substitution mutations in the genes involved in the respiratory chain, even if the found variants were benign or of uncertain meaning. The analysis of mtDNA mutations in the oocyte could provide a better understanding of specific genetic abnormalities and of their possible effect on oocyte developmental competence. This study shows how this approach, based on a massive parallel sequencing of clonally amplified DNA molecules, allows to sequence the entire mitochondrial genome of single oocytes in a short time and with a single analytical run and to verify mtDNA mutations.

Efficacy and safety of follitropin alpha biosimilars compared to their reference product: a Meta-analysis.

AIM: Recently published multicentre, randomized phase III studies suggested the therapeutic equivalence of biosimilar follitropin alpha medicaments compared to the reference product. The aim of this meta-analysis is to pool the results of the three phase III trials in order to provide an overall analysis about the clinical bioequivalence between biosimilars and the originator. METHODS: The studies included in the analysis were pooled together in order to estimate the log odds ratio (OR) for binary outcomes and the weighted mean difference (WMD) for continuous outcomes along with the corresponding 95% confidence intervals (CI) by using a random effects model. The heterogeneity between the studies was evaluated with the Higgins and Chi-square tests. RESULTS: No differences were found in term of number of oocytes retrieved at ovum pick-up, the primary endpoint recommended by the European Medicines Agency. No statistical differences were also found for biochemical pregnancy rate, take home baby rate, total follitropin alpha dose, duration of stimulation, and OHSS risk. A significantly higher clinical pregnancy rate (p = .03) was observed in the originator group in comparison to the biosimilar follitropin alpha. CONCLUSION: Biosimilar follitropin alpha medicaments resulted comparable in comparison to the reference product with respect to the number of oocytes retrieved, that is the primary endpoint recommended by the European Medicines Agency .Further study is needed to evaluate the therapeutic bioequivalence between follitropin alpha biosimilar and the reference medication with respect to secondary endpoints.

Impact of Cigarette Smoking on the Expression of Oxidative Stress-Related Genes in Cumulus Cells Retrieved from Healthy Women Undergoing IVF.

The female reproductive system represents a sensitive target of the harmful effects of cigarette smoke, with folliculogenesis as one of the ovarian processes most affected by this exposure. The aim of this study was to analyze the impact of tobacco smoking on expression of oxidative stress-related genes in cumulus cells (CCs) from smoking and non-smoking women undergoing IVF techniques. Real time PCR technology was used to analyze the gene expression profile of 88 oxidative stress genes enclosed in a 96-well plate array. Statistical significance was assessed by one-way ANOVA. The biological functions and networks/pathways of modulated genes were evidenced by ingenuity pathway analysis software. Promoter methylation analysis was performed by pyrosequencing. Our results showed a down-regulation of 24 genes and an up-regulation of 2 genes (IL6 and SOD2, respectively) involved in defense against oxidative damage, cell cycle regulation, as well as inflammation in CCs from smoking women. IL-6 lower promoter methylation was found in CCs of the smokers group. In conclusion, the disclosed overall downregulation suggests an oxidant-antioxidant imbalance in CCs triggered by cigarette smoking exposure. This evidence adds a piece to the puzzle of the molecular basis of female reproduction and could help underlay the importance of antioxidant treatments for smoking women undergoing IVF protocols.

Successful pregnancy following the transfer of re-vitrified twice-warmed embryos due to the forced cancellation of the primary FET: A case report.

PURPOSE: Embryo cryopreservation represents a central procedure in in-vitro fertilization (IVF) programs. This report documents a Case of a successful pregnancy following the replacement of embryos that had to be re-vitrified due to the forced cancellation of the frozen embryo-transfer (FET). PRINCIPLE RESULTS: The 37- year-old patient was referred to our Assisted Reproductive Technology (ART) unit for idiopathic infertility and recurrent implantation failures. The collection cycle resulted in 8 grade-A cleavage embryos (8-10 blastomeres), that were all vitrified to prevent ovarian hyperstimulation syndrome (OHSS). The first frozen embryo transfer (FET) ended in a biochemical pregnancy and the second in an ectopic pregnancy. In the third attempt, three embryos were warmed but the provider could not complete the transfer due to cervical stenosis. The two surviving embryos were therefore re-vitrified. The final FET attempt, 4 months later, was successful and ended with the live birth of a healthy female baby. CONCLUSIONS: The transfer of re-vitrified twice-warmed embryos may represent a possible option when embryo transfer cannot be performed.

Whole-body exposure to cigarette smoke alters oocyte miRNAs expression in C57BL/6 mice.

Cigarette smoke is toxic for the female reproductive system with particular reference to the ovary. The purpose of the study was to investigate if the microRNAs (miRNAs) pattern could be altered by cigarette smoke exposure in mouse oocytes. For this purpose, C57BL/6 mice were whole-body exposed to three cigarettes daily, 7 days/week, for 2 or 4 months by a specific rodent ventilator. Mice were then superovulated and oocytes collected. MII oocytes pools obtained by single animals were deprived of cumulus cells and used to extract total RNA including miRNAs. TaqMan™ Rodent MicroRNA A Array v2.0 was used to analyze the miRNAs expression profile. The biological functions and the functional networks of the identified up- and downregulated miRNAs were analyzed by ingenuity pathway analysis software. The gene expression of deregulated-miRNAs targets was evaluated. For the first time, the global miRNAs changes in mouse oocyte in response to cigarette smoke exposure were disclosed. Our results revealed significant modulation of miRNAs mainly involved in inflammatory processes, cellular proliferation, and apoptosis. miRNAs expression was altered in a time-dependent manner. Smoke exposure induced an early downregulation of Dicer1. Transcriptional alterations of the modulated miRNAs major targets, estrogen receptor 1, peroxisome proliferator-activated receptor-alpha, and tumor protein 53, as well as that of other key regulatory genes, were evidenced. Cigarette smoke represents a stimulus able to alter miRNAs pattern in mouse oocyte. This study increases our understanding of the ovarian toxicity profile of cigarette smoke, and open new roads toward the identification of biomarkers of oocyte toxicity and dysregulation.

Increased rounds of gonadotropin stimulation have side effects on mouse fallopian tubes and oocytes.

In this study, it was evaluated if increased rounds of gonadotropin stimulation could affect in mice: (i) expression levels of proteins regulating cell cycle and DNA repair in fallopian tubes and (ii) meiotic spindle morphology of ovulated oocytes. To this end, adult female mice were subjected or not (Control) to 6 or 8 rounds of gonadotropin stimulation. Ovulated oocytes were incubated with anti A/B tubulin to evaluate spindle morphology. Fallopian tubes were analyzed to detect Cyclin D1, phospho-p53/p53, phospho-AKT/AKT, phospho-GSK3B/GSK3B, SOX2, OCT3/4, phospho-B-catenin/B-catenin, phospho-CHK1 and phospho-H2A.X protein levels. After 6 rounds, Cyclin D1, p53 and phospho-p53 contents were higher than Control. After 8 rounds, the contents of phosphorylated AKT, GSK3B and p53 as well as of total p53, Cyclin D1 and OCT3/4 significantly increased in comparison with Control. Conversely, SOX2 and B-catenin were similarly expressed among all experimental groups. The finding that phospho-CHK1 and phospho-H2A.X protein levels were undetectable supported the absence of extensive DNA damage. Oocytes number and percentage of normal meiotic spindles drastically decreased from 6 rounds onward. Altogether, our results demonstrated that 6 and 8 cycles of gonadotropin stimulation reduce mouse reproductive performances by inducing over-expression and over-activation of proteins controlling cell cycle progression in fallopian tubes and by impairing oocyte spindle.

Folate serum levels in Italian women entering an in vitro fertilization program.

Italian public health authorities recommend women of childbearing age to assume a daily dose of 0.4 mg of folic acid (FA) from at least one month before conception in order to reduce the risk of having children affected by neural tube defects (NTDs). In this study, folate, homocysteine and vitamin B12 serum levels were determined in 77 women entering an in vitro fertilization program. About 75% of patients had serum folate values compatible with the intake of the recommended dose of FA for at least three months, whereas only the 61% of them reached or exceeded the serum folate concentration regarded as the optimal concentration during the periconceptional period. Mean vitamin B12 serum levels and mean homocysteine plasma levels resulted in normal range in all the women with mean values of 381.2 ± 2.2 pg/ml and 8.48 ± 2.2 µmol/l, respectively. In conclusion, only a portion of women entering an IVF program presents proper folate levels.

Clinical experience with an ovarian stimulation protocol for intrauterine insemination adopting a gonadotropin releasing hormone antagonist at low dose.

Studies testing the effectiveness of GnRH antagonists in controlled ovarian stimulation (COS) for intrauterine insemination (IUI) have provided controversial results. The present study was undertaken to evaluate, whether the use of a half of the conventional dose of the GnRH antagonist cetrorelix can be effective in increasing the successful rate of IUI cycles. Patients started COS with human menopausal gonadotropin (hMG) on day three of the menstrual cycle. Cetrorelix was started when at least one follicle of ≥14 mm, was detected at the ultrasound scan, according to the flexible multiple daily dose protocol, and continued until the trigger day with recombinant hCG. Patients adopting GnRH antagonist at low dose had a pregnancy rate (21.7%) that was significantly higher (p < 0.05) in comparison to women receiving hMG only (8.7%). These results suggest that adding a reduced dose of GnRH antagonist to the COS for IUI cycles significantly improves the outcome of the procedure.

Cigarette smoke is associated with altered expression of antioxidant enzymes in granulosa cells from women undergoing in vitro fertilization.

This study was undertaken to evaluate whether cigarette smoke is associated with changes in the expression of antioxidant enzymes in granulosa cells of women undergoing IVF treatments. For this aim, the expression of three antioxidant enzymes (SOD1, SOD2 and catalase) in non-smokers (n = 20) and smokers (n = 20) was analyzed. There was a statistically significant overexpression of SOD2 and catalase mRNA levels in smokers in comparison with non-smokers. Cigarette smoking was associated with a lower fertilization rate, implantation rate and pregnancy rate in comparison with non-smokers. There was no effect on retrieved oocytes number, metaphase II oocytes number, quality of embryos transferred and live birth rate. These findings suggest that cigarette smoke initiates oxidative stress in granulosa cells.

Thrombophilia and outcomes of assisted reproduction technologies: a systematic review and meta-analysis.

Thrombophilia has been associated with pregnancy complications and recurrent miscarriage. The aim of this systematic review was to evaluate the controversial association between thrombophilia and failures of assisted reproduction technology (ART). A systematic search of the literature for studies reporting on thrombophilia in women undergoing ART up to April 2011 yielded 33 studies (23 evaluating anti-phospholipid antibodies, 5 inherited thrombophilia, and 5 both) involving 6092 patients. Overall, methodologic quality of the studies was poor. Combined results from case-control studies showed that factor V Leiden was significantly more prevalent among women with ART failure compared with fertile parous women or those achieving pregnancy after ART (odds ratio = 3.08; 95% confidence interval, 1.77-5.36). The prothrombin mutation, methylenetetrahydrofolate reductase mutation, deficiency of protein S, protein C, or anti-thrombin were all not associated with ART failure. Women with ART failure tested more frequently positive for anti-phospholipids antibodies (odds ratio = 3.33; 95% confidence interval, 1.77-6.26) with evidence of high degree of between-study heterogeneity (I(2) = 75%; P < .00001). Prospective cohort studies did not show significant associations between thrombophilia and ART outcomes. Although case-control studies suggest that women experiencing ART failures are more frequently positive for factor V Leiden and anti-phospholipid antibodies, the evidence is inconclusive and not supported by cohort studies.

Nutrition, female fertility and in vitro fertilization outcomes.

The investigation of modifiable factors that may exert influences on female reproductive health and in vitro fertilization (IVF) outcomes is increasing. Growing attention is being paid to nutrition. The aim of the present review is to recapitulate the current understanding on the effects of nutrition on female fertility and IVF outcomes. In particular, the three main classes of macromolecules have been analysed i.e. carbohydrates, proteins and fatty acids. An increasing number of studies have focused on the potential benefit of whole grain, vegetables and omega-3 polynsatured fatty acids (ω-3 PUFAs) on reproductive outcomes. Controversial results exist regarding the consumption of omega-6 (ω-6) PUFAs and dairy. Overall, nutrition appears to represent a modifiable factor that may play a significant role in the context of female reproduction and IVF outcomes, but the limited number of studies and the discrepancies between the available data call for further research in the area.

The fungicide mancozeb induces toxic effects on mammalian granulosa cells.

The ethylene-bis-dithiocarbamate mancozeb is a widely used fungicide with low reported toxicity in mammals. In mice, mancozeb induces embryo apoptosis, affects oocyte meiotic spindle morphology and impairs fertilization rate even when used at very low concentrations. We evaluated the toxic effects of mancozeb on the mouse and human ovarian somatic granulosa cells. We examined parameters such as cell morphology, induction of apoptosis, and p53 expression levels. Mouse granulosa cells exposed to mancozeb underwent a time- and dose-dependent modification of their morphology, and acquired the ability to migrate but not to proliferate. The expression level of p53, in terms of mRNA and protein content, decreased significantly in comparison with unexposed cells, but no change in apoptosis was recorded. Toxic effects could be attributed, at least in part, to the presence of ethylenthiourea (ETU), the main mancozeb catabolite, which was found in culture medium. Human granulosa cells also showed dose-dependent morphological changes and reduced p53 expression levels after exposure to mancozeb. Altogether, these results indicate that mancozeb affects the somatic cells of the mammalian ovarian follicles by inducing a premalignant-like status, and that such damage occurs to the same extent in both mouse and human GC. These results further substantiate the concept that mancozeb should be regarded as a reproductive toxicant.

Assisted reproduction outcomes after embryo transfers requiring a malleable stylet.

OBJECTIVE: This retrospective case-control study was undertaken to assess the impact of a malleable stylet application on embryo transfer (ET) outcome. METHODS: The Wallace malleable stylet was used when the soft inner catheter could not negotiate the internal os during ultrasound-guided ET. Patients having an age ≤37 years, with three embryos replaced were included in the analysis. Implantation rate, clinical pregnancy rate and live birth rate were the main outcome measures. RESULTS: The Wallace malleable stylet was applied in 164 (29.6%) of the 553 ETs evaluated. Implantation rate, clinical pregnancy rate and live birth rate showed a 5.6% (19.4% vs. 13.8%), a 10.8% (41.9% vs. 31.1%) and a 9.9% (37.3% vs. 27.4%) absolute difference, respectively, in favor of ETs not requiring the stylet. These differences were statistically significant (P < 0.05). CONCLUSIONS: ETs requiring stylet are associated with lower implantation, clinical pregnancy and live birth rates.

The Aftermath of Long-Term Cigarette Smoking on Telomere Length and Mitochondrial DNA Copy Number in Human Cumulus Cells Prior to In Vitro Fertilization-A Pilot Study.

Cigarette smoking among women of reproductive age is known to take a toll on systemic health and fertility potential by severely impacting ovarian tissues and cells, such as granulosa and cumulus cells (CCs). The purpose of this study was to determine the potential damage caused by tobacco smoke at a molecular level in the CCs of females who had undergone in vitro fertilization. The level of intracellular damage was determined by estimating the average telomere length (TL) and mitochondrial DNA copy number (mtDNA-CN), as well as the expression profile of telomere maintenance genes TERF1, TERF2, POT1 and microRNAs miR-155, miR-23a and miR-185. Western blotting analysis was performed to detect consequent protein levels of TERF1, TERF2 and POT1. Our results evidenced significantly lower relative TL and mtDNA-CN and a down-regulation pattern for all three described genes and corresponding proteins in the CCs of smokers compared with controls (p < 0.05). No significant differences were found in the miRNAs' modulation. Combined, our data add another piece to the puzzle of the complex regulatory molecular networks controlling the general effects of tobacco smoke in CCs. This pilot study extends the until now modest number of studies simultaneously investigating the mtDNA-CN and TL pathways in the human CCs of smoking women.

Cytogenetic findings and reproductive outcome of infertile couples referred to an assisted reproduction program.

It is still undefined whether all the couples entering an assisted reproduction program should undergo to karyotype analysis. The present study was conducted to determine the prevalence of chromosomal abnormalities in a non-selected sample of 1,146 couples referred to assisted reproduction technologies (ART), and to analyze the outcome of pregnancies from couples in whom cytogenetic anomalies were detected. Irrespective of the infertility factor, fertilization was achieved by intracytoplasmic sperm injection (ICSI). A total number of 35 karyotype anomalies were diagnosed, corresponding to an abnormality frequency of 1.52% (1.83% for men and 1.22% for women). As could be expected, the majority of men presenting karyotype anomalies had a low sperm count. Among women, the majority of cytogenetic anomalies were detected in individual not presenting risk factors for aberrant karyotype. Around 41% of pregnancies achieved in couples presenting chromosomal anomalies ended in spontaneous abortion. Information on fetal karyotype was limited. No major malformations were observed among newborns from parents with abnormal karyotype. In consideration of the elevated frequency of pregnancy loss, it seems advisable to recommend that chromosomal analysis be performed in all couples undergoing ART. This with the aim of identifying patients that would possibly benefit from pre-implantation genetic diagnosis.

Effectiveness of a low gonadotrophin-releasing hormone antagonist dose in preventing premature luteinizing hormone rise during controlled ovarian stimulation.

The current prospective randomized study was designed to test the efficacy of a low dose (0.125 mg/day) of the gonadotrophin-releasing hormone antagonist cetrorelix in preventing premature luteinizing hormone (LH) rise during controlled ovarian stimulation in comparison with the standard dose of 0.25 mg/day. Ovarian stimulation was started with 225 IU of recombinant follicle stimulating hormone (FSH) on day 2 of the menstrual cycle. Cetrorelix was injected daily from day 6 of gonadotropin administration. Blood was sampled from each woman on day 3 of ovarian stimulation and then daily from day 5 onward up to human chorionic gonadotropin administration for analysis of FSH, LH, progesterone, and estradiol. LH rise was defined as serum LH ≥ 10 mIU/ml. There were 40 patients receiving cetrorelix at 0.25 mg/day and 36 patients receiving cetrorelix at 0.125 mg/day. Premature LH rise was recorded in 10% of patients injecting antagonist at 0.25 mg/day and in 14% of patients administered with antagonist at 0.125 mg/day. These frequencies did not differ statistically. In conclusion, our results suggest that a cetrorelix dose of 0.125 mg/day is effective as the standard dose (0.25 mg/day) in preventing premature LH rise during controlled ovarian stimulation.

Impact of a single endometrial injury on assisted reproductive technology outcome: a preliminary observational study.

OBJECTIVE: To find a more acceptable strategy for our patients to improve endometrial receptivity in assisted reproductive technologies (ART) and to study the impact of a single endometrial sampling performed on day 21 of the spontaneous menstrual cycle. STUDY DESIGN: In this preliminary study, the possible beneficial effects on ART outcome of a single biopsy performed on the midluteal phase (day 21) of the spontaneous menstrual cycle preceding controlled ovarian stimulation was tested in a group of patients with 3 or more implantation failures. RESULTS: After endometrial biopsy 45.94% of patients with more than two failed ART cycles obtained clinical pregnancy. CONCLUSION: Although these preliminary results are encouraging, further investigation is needed to validate the study.

Novel Insights on the Role of Nitric Oxide in the Ovary: A Review of the Literature.

Nitric oxide (NO) is formed during the oxidation of L-arginine to L-citrulline by the action of multiple isoenzymes of NO synthase (NOS): neuronal NOS (nNOS), endotelial NOS (eNOS), and inducible NOS (iNOS). NO plays a relevant role in the vascular endothelium, in central and peripheral neurons, and in immunity and inflammatory systems. In addition, several authors showed a consistent contribution of NO to different aspects of the reproductive physiology. The aim of the present review is to analyse the published data on the role of NO within the ovary. It has been demonstrated that the multiple isoenzymes of NOS are expressed and localized in the ovary of different species. More to the point, a consistent role was ascribed to NO in the processes of steroidogenesis, folliculogenesis, and oocyte meiotic maturation in in vitro and in vivo studies using animal models. Unfortunately, there are few nitric oxide data for humans; there are preliminary data on the implication of nitric oxide for oocyte/embryo quality and in-vitro fertilization/embryo transfer (IVF/ET) parameters. NO plays a remarkable role in the ovary, but more investigation is needed, in particular in the context of human ovarian physiology.

In Vivo Cigarette Smoke Exposure to Examine the Expression of Genes Involved in the Inflammatory Response in the Mouse Uterus.

Cigarette smoke may impair uterine function, but the underlying mechanisms are poorly characterized. In this article, we describe the methodology for whole-body exposure to cigarette smoke together with assessment of the impact of this exposure on the expression of a panel of genes related to stress and toxicity pathways in mouse uteri using an in vivo model. C57BL/6 mice are whole-body-exposed to three cigarettes daily, 7 days/week, for 2 months using a specific rodent ventilator. Uteri are then collected and subjected to qRT-PCR analysis using the Stress & Toxicity PathwayFinder RT2 Profiler PCR Array (Qiagen). Cigarette smoke was found to be associated with an upregulation (≥2-fold) of C-reactive protein (Crp; 2.65-fold, p-value = 0.02), growth arrest and DNA-damage-inducible45γ (Gadd45γ; 2.11-fold, p-value = 0.04), interferon γ (Ifnγ; 2.05-fold, p-value = 0.01), and interleukin1α (Il1α; 7.74-fold, p-value = 0.003) and downregulation of matrix metallopeptidase-9 (Mmp9; -2.42-fold, p-value = 0.01). The protocol used in this study may represent a new experimental model of mouse in vivo mainstream exposure to cigarette smoke. In addition, the resulting overexpression of pro-inflammatory cytokines and genes involved in cell cycle proliferation, together with the downregulation of extracellular matrix metallopeptidases, may represent a toxicological response to cigarette smoke exposure, with potential repercussion for the processes of uterine remodeling and growth that are essential for uterine receptiveness. A recommendation to expand upon this research area is made. © 2021 Wiley Periodicals LLC.

Maternal use of fluconazole and congenital malformations in the progeny: A meta-analysis of the literature.

Fluconazole is a bis-triazole agent used in the treatment of superficial and systemic fungal infections, with vaginal candidiasis being one of the commonest indications to fluconazole treatment. There is increasing concern regarding the teratogenic potential of fluconazole. The aim of this meta-analysis is to pool the literature data in order to evaluate the possible association between fluconazole exposure during pregnancy and birth defects. A total of nine studies were included in the meta-analysis. Results were expressed as odds ratios (OR) with 95 % confidence intervals (CI) and statistical heterogeneity between the studies was evaluated with Higgins index (I2) and Q-test (Q). A p-value < 0.05 referred to the effect was considered statistically significant. The maternal exposure to fluconazole during the first trimester of pregnancy is correlated with increased prevalence of heart defects in the offspring for both low dose (OR 1.95, 95 % CI 1.18-3.21; P = 0.01) and any dose (OR 1.79, 95 % CI 1.18-2.71; P = 0.01). No association was found between gestational exposure to fluconazole and increased risk of spontaneous abortion or stillbirth. Fluconazole should be regarded as a human teratogen and should be cautiously prescribed to pregnant women and to women of childbearing potential.