Exploring the interaction of N-(benzothiazol-2-yl)pyrrolamide DNA gyrase inhibitors with the GyrB ATP-binding site lipophilic floor: A medicinal chemistry and QTAIM study.

N-(Benzothiazole-2-yl)pyrrolamide DNA gyrase inhibitors with benzyl or phenethyl substituents attached to position 3 of the benzothiazole ring or to the carboxamide nitrogen atom were prepared and studied for their inhibition of Escherichia coli DNA gyrase by supercoiling assay. Compared to inhibitors bearing the substituents at position 4 of the benzothiazole ring, the inhibition was attenuated by moving the substituent to position 3 and further to the carboxamide nitrogen atom. A co-crystal structure of (Z)-3-benzyl-2-((4,5-dibromo-1H-pyrrole-2-carbonyl)imino)-2,3-dihydrobenzo[d]-thiazole-6-carboxylic acid (I) in complex with E. coli GyrB24 (ATPase subdomain) was solved, revealing the binding mode of this type of inhibitor to the ATP-binding pocket of the E. coli GyrB subunit. The key binding interactions were identified and their contribution to binding was rationalised by quantum theory of atoms in molecules (QTAIM) analysis. Our study shows that the benzyl or phenethyl substituents bound to the benzothiazole core interact with the lipophilic floor of the active site, which consists mainly of residues Gly101, Gly102, Lys103 and Ser108. Compounds with substituents at position 3 of the benzothiazole core were up to two orders of magnitude more effective than compounds with substituents at the carboxamide nitrogen. In addition, the 6-oxalylamino compounds were more potent inhibitors of E. coli DNA gyrase than the corresponding 6-acetamido analogues.

The A domain of clonal complex 1-type fibronectin binding protein B promotes adherence and biofilm formation in Staphylococcus aureus.

Adhesive interactions between Staphylococcus aureus and the host rely on cell-wall-anchored proteins such as fibronectin-binding protein B (FnBPB). Recently we showed that the FnBPB protein expressed by clonal complex (CC) 1 isolates of S. aureus mediates bacterial adhesion to corneodesmosin. The proposed ligand-binding region of CC1-type FnBPB shares just 60 % amino acid identity with the archetypal FnBPB protein from CC8. Here we investigated ligand binding and biofilm formation by CC1-type FnBPB. We found that the A domain of FnBPB binds to fibrinogen and corneodesmosin and identified residues within the hydrophobic ligand trench in the A domain that are essential for the binding of CC1-type FnBPB to ligands and during biofilm formation. We further investigated the interplay between different ligands and the influence of ligand binding on biofilm formation. Overall, our study provides new insights into the requirements for CC1-type FnBPB-mediated adhesion to host proteins and FnBPB-mediated biofilm formation in S. aureus.

Q Chat Space: Assessing the Feasibility and Acceptability of an Internet-Based Support Program for LGBTQ Youth.

There are few psychosocial support programs specifically designed to meet the unique developmental and health needs of LGBTQ youth. Even when available, many youth face significant barriers to accessing LGBTQ-specific services for fear of being "outed" to parents, peers, and community members. The current study assessed the utility, feasibility, and acceptability of a synchronous, adult-facilitated, chat-based Internet community support program for LGBTQ youth aged 13-19. Chat transcripts were analyzed to examine how LGBTQ youth used the chat-based platform to connect with peers and trusted adults. A separate user satisfaction survey was collected to assess the personal (e.g., sexual orientation, gender identity, age) and contextual (e.g., geography, family environment) characteristics of youth engaging in the platform, their preferred topics of discussion, and their satisfaction with the program focus and facilitators. Qualitative data analysis demonstrated the degree to which LGBTQ youth were comfortable disclosing difficult and challenging situations with family, friends, and in their community and in seeking support from peers and facilitators online. Youth also used the platform to explore facets of sexual and gender identity/expression and self-acceptance. Overall, users were very satisfied with the platform, and participants accurately reflect the program's desired populations for engagement (e.g., LGBTQ youth of color, LGBTQ youth in the South). Together, findings support the feasibility and acceptability of synchronous, adult-facilitated, chat-based Internet programs to connect and support LGBTQ youth, which encourage future research and innovation in service delivery.

Structural and mechanistic analysis of ATPase inhibitors targeting mycobacterial DNA gyrase.

OBJECTIVES: To evaluate the efficacy of two novel compounds against mycobacteria and determine the molecular basis of their action on DNA gyrase using structural and mechanistic approaches. METHODS: Redx03863 and Redx04739 were tested in antibacterial assays, and also against their target, DNA gyrase, using DNA supercoiling and ATPase assays. X-ray crystallography was used to determine the structure of the gyrase B protein ATPase sub-domain from Mycobacterium smegmatis complexed with the aminocoumarin drug novobiocin, and structures of the same domain from Mycobacterium thermoresistibile complexed with novobiocin, and also with Redx03863. RESULTS: Both compounds, Redx03863 and Redx04739, were active against selected Gram-positive and Gram-negative species, with Redx03863 being the more potent, and Redx04739 showing selectivity against M. smegmatis. Both compounds were potent inhibitors of the supercoiling and ATPase reactions of DNA gyrase, but did not appreciably affect the ATP-independent relaxation reaction. The structure of Redx03863 bound to the gyrase B protein ATPase sub-domain from M. thermoresistibile shows that it binds at a site adjacent to the ATP- and novobiocin-binding sites. We found that most of the mutations that we made in the Redx03863-binding pocket, based on the structure, rendered gyrase inactive. CONCLUSIONS: Redx03863 and Redx04739 inhibit gyrase by preventing the binding of ATP. The fact that the Redx03863-binding pocket is distinct from that of novobiocin, coupled with the lack of activity of resistant mutants, suggests that such compounds could have potential to be further exploited as antibiotics.

Creating Systems-Level Change to Better Support Expectant and Parenting Young People: A Case Study.

INTRODUCTION: Expectant and parenting young people (young parents) require diverse services to support their health, educational success, and family functioning. Rarely can the needs of young parents be met by a single school or service provider. This case study examines how one large school district funded through the pathways to success initiative was able to facilitate systems change to increase young parents' access to and use of supportive services. METHODS: Data sources include a needs and resources assessment, quarterly reports documenting grantee effort, sustainability plans, social network analysis, and capstone interviews. All data sources were systematically reviewed to identify the existing context prior to the start of the initiative, the changes that resulted from the initiative, and efforts that could potentially be maintained beyond the grant period. RESULTS: The community context prior to Pathways implementation was one of disconnected services and missed opportunities. The full-time program coordinator hired by the district focused on systems-level change and facilitated connections between organizations. This greater connectivity contributed to increased collaboration with the goal of producing lasting benefits for young parents. DISCUSSION: Promoting sustainable connections and collaboration at the systems level can help dismantle barriers to service access and benefit young parents.

Fresh transfer of Day 5 slow-growing embryos versus deferred transfer of vitrified, fully expanded Day 6 blastocysts: which is the optimal approach?

STUDY QUESTION: In IVF cycles in which the entire embryo cohort is slow growing, is it optimal to perform fresh transfer in Day 5 or to extend the culture and transfer in subsequent vitrified-warmed cycles? SUMMARY ANSWER: The outcomes depend on the degree of embryo development on Day 5. WHAT IS KNOWN ALREADY: Slow-growing blastocysts have lower implantation potential when transferred in fresh cycles. It has been suggested that embryo-endometrial asynchrony could explain this finding. However, studies that compared Days 5 and 6 embryos in frozen embryo transfer (FET) cycles showed contradictory results. There is still a lack of evidence regarding the best approach, performing fresh transfer or deferring transfer and continuing culture until fully developed blastocysts are achieved, when the entire cohort of embryos is slow growing. STUDY DESIGN SIZE, DURATION: This was a retrospective study that included 477 women aged <40 years who underwent fresh Day 5 single embryo transfer of slow-growing embryos and subsequent FET cycles of fully expanded blastocysts (FEB) originating from the same IVF cycle between 2012 and 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study included cycles in which the embryos either began blastulation by Day 5 of culture but did not reach the fully expanded stage (Gardner Stage III) or had delayed blastulation with only morula embryos present by Day 5 of culture. All of the subjects in the study underwent elective, single embryo transfer (slow or delayed blastocysts) on Day 5 and had at least one embryo that developed into a FEB on extended culture Day 6 that was suitable for vitrification. All subjects, regardless of the outcome of the fresh transfer, returned for at least one subsequent FET cycle of Day 6 embryos. MAIN RESULTS AND ROLE OF CHANCE: A total of 1070 embryo transfer cycles (fresh + FET) were included. Of them, 365 women had elective, fresh, single transfer of slow-growing blastocysts (Group I) and 112 had elective, fresh, single morula transfer (Group II). Groups I and II underwent a subsequent 457 and 136 FET cycles, respectively. The mean age of Group I was 33.8 ± 2.9 years, the proportion of Day 5 embryos that developed to FEB by Day 6 was 92%, and the number of blastocysts vitrified was 627 (average of 1.71 blastocysts per cycle). The outcomes of fresh and FET cycles were comparable regarding clinical pregnancy rate (CPR) (31.0 vs. 30.4%, P = 0.86) and live birth rate (LBR) (23.3 vs. 20.3%, P = 0.15). In Group II, the mean age was 35.8 ± 3.4 years and the proportion of morula embryos that developed to FEB by Day 6 was 72%. The number of blastocysts vitrified on Day 6 was 155 (1.38 per cycle). The transfer of fresh embryos in Group II resulted in significantly lower clinical pregnancy (5.3 vs. 30.1%, P < 0.001) and LBRs (1.8 vs. 20.5%, P < 0.001). The results did not change after controlling for possible confounding factors. LIMITATIONS AND REASONS FOR CAUTION: The retrospective design of the study is a major limitation. Although we compared the outcomes of embryos that originated from the same cohort, the FET cycles could have been overrepresented by older patients and those with poorer prognoses. Furthermore, the study included only cycles in which there were blastocysts available for cryopreservation on Day 6; therefore, the results were not be applicable for those who had mandatory Day 5 transfer with no embryos available for vitrification. WIDER IMPLICATIONS OF THE FINDINGS: Fresh transfer of embryos that begin blastulation on Day 5 results in similar outcomes to the transfer of FEB originating from the same cohort. However, in cases where only morula/compacting embryos are available by Day 5, extending culture until FEB are achieved and then performing subsequent FET will result in significantly higher LBRs. STUDY FUNDING/COMPETING INTEREST(S): No external funding was used for this study. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Relationship between pre-ICSI meiotic spindle angle, ovarian reserve, gonadotropin stimulation, and pregnancy outcomes.

PURPOSE: The goals of this study are to analyze the clinical pregnancy rate as a function of the pre-intracytoplasmic sperm injection (ICSI) oocyte spindle angle and determine factors which can be associated with different spindle angles, if clinically relevant. METHODS: Fifty-eight patients, who underwent their first ICSI cycle from January to December 2013, were included. Eight hundred thirty oocytes were collected, and 648 were metaphase II (MII) on retrieval day. Spindles were characterized in terms of visibility and position in relation to the first polar body (PB). Oocytes were separated into four groups based on angle: (group 1, n = 297) 0°-29°; (group 2 n = 212) 30°-89°; (group 3, n = 72) ≥90°; and those with no visible spindle (group 4, n = 67). RESULTS: The rate of blastocyst development was associated with the spindle angle (p = 0.002). The rate of good quality blastocysts were as follows: group 1 (42%), group 2 (30%), group 3 (35%), and group 4 (19%) (p = 0.02). Pregnancy and live birth rates were also affected (p = 0.007 and p = 0.046, respectively). Antral follicle count (AFC) (p = 0.001), total FSH stimulating dose (p = 0.0001), and peak serum estradiol level (p = 0.0001) were associated with spindle angle grouping. Miscarriage rates trended different (p = 0.07). On the other hand, day 3 follicle-stimulating hormone (FSH) levels and female and male age were not associated with spindle angle grouping. CONCLUSIONS: Embryos resulting from oocytes with pre-ICSI spindle angles between 0° and 29° were associated with better blastocyst, pregnancy, live birth, and miscarriage rates when compared to oocytes that had no visible spindle. Low ovarian reserve and excessive stimulation were also associated with lack of spindle and therefore lower pregnancy outcomes.

Profound reduction in tamoxifen active metabolite endoxifen in a breast cancer patient treated with rifampin prior to initiation of an anti-TNFα biologic for ulcerative colitis: a case report.

BACKGROUND: Tamoxifen, a common anti-estrogen breast cancer medication, is a prodrug that undergoes bioactivation via cytochrome P450 enzymes, CYP2D6 and to a lesser degree, CYP3A4 to form the active metabolite endoxifen. With an increasing use of oral anti-cancer drugs, the risk for drug-drug interactions mediated by enzyme inhibitors and inducers may also be expected to increase. Here we report the first case demonstrating a potent drug-drug interaction in a real-world clinical setting between tamoxifen and rifampin in a breast cancer patient being treated concurrently for ulcerative colitis. CASE PRESENTATION: We describe a patient on adjuvant tamoxifen therapy for breast cancer that was prescribed rifampin for TB prophylaxis prior to initiation of an anti-tumor necrosis factor (TNF)-α agent due to worsening ulcerative colitis. This 39 year old Caucasian woman had been followed by our personalized medicine clinic where CYP2D6 genotyping and therapeutic monitoring of tamoxifen and endoxifen levels had been carried out. The patient, known to be a CYP2D6 intermediate metabolizer, had a previous history of therapeutic endoxifen levels. Upon admission to hospital for a major flare of her ulcerative colitis a clinical decision was made to initiate an anti-TNFα biological agent. Due to concerns regarding latent TB, rifampin as an anti-mycobacterial agent was initiated which the patient was only able tolerate for 10 days. Interestingly, her plasma endoxifen concentration measured 2 weeks after cessation of rifampin was sub-therapeutic at 15.8 nM and well below her previous endoxifen levels which exceeded 40 nM. CONCLUSION: Rifampin should be avoided in patients on tamoxifen therapy for breast cancer unless continued tamoxifen efficacy can be assured through endoxifen monitoring. Drug-drug interactions can pose a significant risk of sub-therapeutic benefit in tamoxifen patients.

Human platelets contain a pool of free zinc in dense granules.

Background: Activated platelets release procoagulant factors that include Ca2+ and Zn2+. Releasable Ca2+ stores have been identified in platelet dense granules and the dense tubular system, but similar stores of free Zn2+ have not been identified. Objectives: Guided by studies of platelet Ca2+, we employed minimally disruptive methods to identify and localize concentrated free Zn2+ in human platelets. Methods: Resting platelets from normal donors (NDs), patients with gray platelet syndrome (GPS) lacking α-granules, and patients with Hermansky-Pudlak syndrome (HPS) deficient in dense granules were loaded with cell-permeant fluorescent probes specific to free Ca2+ or Zn2+. Ion concentrations were detected in fixed cells as bright puncta via high-resolution confocal microscopy. Ions were also directly detected via transmission electron microscopy and energy dispersive X-ray analysis. Levels of total platelet Ca, Zn, and Mg were measured via inductively coupled plasma optical emission spectroscopy. Results: Fluorescent Zn2+ puncta counts were similar in ND and GPS platelets and markedly lower in HPS platelets, pointing to dense granules as likely reservoirs of free Zn2+. This localization was supported by direct detection of Ca2+, Zn2+, and Na+ in platelet dense granules via transmission electron microscopy and energy dispersive X-ray analysis. Measurements of total platelet Ca, Zn, and Mg via inductively coupled plasma optical emission spectroscopy indicated that free Zn2+ represents a small proportion of total platelet zinc, consistent with the strong affinity of Zn2+ for binding proteins, including several abundant in platelet α-granules. Conclusion: We conclude that normal human platelets contain a pool of free Zn2+ concentrated in dense granules that is available for secretion upon platelet activation and potentially contributes to hemostasis.

Spindle examination in unfertilized eggs using the polarization microscope can assist rescue ICSI.

Rescue ICSI can induce a high rate of 3 pronuclei (PN) formation from double insemination in eggs already fertilized by IVF but lacking signs of normal pronuclear formation. This study was performed to determine whether the number of 3PN embryos could be reduced by using the polarization microscope for rescue intracytoplasmic sperm injection (ICSI). As a study group, after conventional insemination, 81 unfertilized mature oocytes from 11 couples were checked for the number of spindles using the polarization microscope. One spindle (82.7%) or two spindles (17.3%) were observed in this group. Rescue ICSI was only performed on the unfertilized oocytes showing one spindle. In the control group, 87 mature oocytes which lacked visualization of any fertilization signs were selected for rescue ICSI and none of them underwent observation of the spindle. After rescue ICSI, the normal fertilization rate in the study group was significantly higher than in the control (68.7% versus 43.7%; P=0.0032). The rate of 3PN or 4PN embryos was significantly decreased in the study group with one spindle compared with the group without observation of the spindle (4.5% versus 26.4%; P=0.0004).

The Perceived Health Implications of Policies and Rhetoric Targeting Transgender and Gender Diverse Youth: A Community-Based Qualitative Study.

Purpose: In early 2021, >50 bills targeting transgender and gender diverse (TGD) youth in the United States were proposed; these policies and the rhetoric surrounding them is associated with TGD health disparities. Methods: A community-based qualitative study utilized focus groups with a TGD youth research advisory board to explore their knowledge and perceived impacts of the current policy climate and rhetoric in one Midwestern state. Results: Themes revealed (1) mental health, (2) structural impacts, and (3) messages to policymakers. Conclusions: Discriminatory policies and rhetoric harm TGD youth; health professionals should denounce the harmful disinformation perpetuated by these policies.

Exploring the 5-Substituted 2-Aminobenzothiazole-Based DNA Gyrase B Inhibitors Active against ESKAPE Pathogens.

We present a new series of 2-aminobenzothiazole-based DNA gyrase B inhibitors with promising activity against ESKAPE bacterial pathogens. Based on the binding information extracted from the cocrystal structure of DNA gyrase B inhibitor A, in complex with Escherichia coli GyrB24, we expanded the chemical space of the benzothiazole-based series to the C5 position of the benzothiazole ring. In particular, compound E showed low nanomolar inhibition of DNA gyrase (IC50 < 10 nM) and broad-spectrum antibacterial activity against pathogens belonging to the ESKAPE group, with the minimum inhibitory concentration < 0.03 µg/mL for most Gram-positive strains and 4-16 µg/mL against Gram-negative E. coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. To understand the binding mode of the synthesized inhibitors, a combination of docking calculations, molecular dynamics (MD) simulations, and MD-derived structure-based pharmacophore modeling was performed. The computational analysis has revealed that the substitution at position C5 can be used to modify the physicochemical properties and antibacterial spectrum and enhance the inhibitory potency of the compounds. Additionally, a discussion of challenges associated with the synthesis of 5-substituted 2-aminobenzothiazoles is presented.

Discovery and Hit-to-Lead Optimization of Benzothiazole Scaffold-Based DNA Gyrase Inhibitors with Potent Activity against Acinetobacter baumannii and Pseudomonas aeruginosa.

We have developed compounds with a promising activity against Acinetobacter baumannii and Pseudomonas aeruginosa, which are both on the WHO priority list of antibiotic-resistant bacteria. Starting from DNA gyrase inhibitor 1, we identified compound 27, featuring a 10-fold improved aqueous solubility, a 10-fold improved inhibition of topoisomerase IV from A. baumannii and P. aeruginosa, a 10-fold decreased inhibition of human topoisomerase IIα, and no cross-resistance to novobiocin. Cocrystal structures of 1 in complex with Escherichia coli GyrB24 and (S)-27 in complex with A. baumannii GyrB23 and P. aeruginosa GyrB24 revealed their binding to the ATP-binding pocket of the GyrB subunit. In further optimization steps, solubility, plasma free fraction, and other ADME properties of 27 were improved by fine-tuning of lipophilicity. In particular, analogs of 27 with retained anti-Gram-negative activity and improved plasma free fraction were identified. The series was found to be nongenotoxic, nonmutagenic, devoid of mitochondrial toxicity, and possessed no ion channel liabilities.

OXA-66 structure and oligomerisation of OXAAb enzymes.

The OXA ß-lactamases are responsible for hydrolysing ß-lactam antibiotics and contribute to the multidrug-resistant phenotype of several major human pathogens. The OXAAb enzymes are intrinsic to Acinetobacter baumannii and can confer resistance to carbapenem antibiotics. Here we determined the structure of the most prevalent OXAAb enzyme, OXA-66. The structure of OXA-66 was solved at a resolution of 2.1 Å and found to be very similar to the structure of OXA-51, the only other OXAAb enzyme that has had its structure solved. Our data contained one molecule per asymmetric unit, and analysis of positions responsible for dimer formation in other OXA enzymes suggest that OXA-66 likely exists as a monomer.

Influence of Maternal Age and Ovarian Reserve on the Decision to Continue or to Cancel IVF Cycles in Patients with One or Two Large Follicles: a Dual Effect.

This study aimed to study whether IVF stimulation that results in one or two mature follicles should proceed to oocyte retrieval. This is a retrospective cohort study conducted at McGill University Health Center on 459 patients who underwent IVF treatment between 2011 and 2014, undergoing hormonal stimulation and monitoring of their ovarian response. The primary outcomes were pregnancy and live birth rates. Statistical modeling was used to determine individual roles of patient age and ovarian reserve on outcomes, while controlling for the other factors. Of the 459 cycles included in the study, 360 cycles (78.4%) ended in embryo transfer. Live birth rates per cycle were 15.6%, for the ≤ 34-year-olds; 6.5%, for the 35-39-year-olds; and 2.7%, for the ≥ 40-year-olds (p < 0.01). Twenty-five percent of the cycles in the ≥ 40-year-old group were canceled versus 17% and 15% in the 35-39-year-old and ≤ 34-year-old groups, respectively (p < 0.05). Testing likelihood of live birth as a function of age and antral follicular count (AFC) revealed that a 1-year increase in age reduces the likelihood of live birth by 11% (p < 0.05) and one-unit increase in AFC count leads to a 9% increase in the odds of a live birth (p < 0.05). For the youngest age group, the AFC had a most significant effect, and those with AFC > 11 had 56% live birth rate, while those with AFC ≤ 11 had only 6% of live birth rate. This study supports a shift in reasoning from age being the predictor of outcomes in women with a low response at IVF to both age and ovarian reserve needing to be taken into consideration.

The crystal structure of mycobacterial epoxide hydrolase A.

The human pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis resulting in over 1 million fatalities every year, despite decades of research into the development of new anti-TB compounds. Unlike most other organisms M. tuberculosis has six putative genes for epoxide hydrolases (EH) of the α/ß-hydrolase family with little known about their individual substrates, suggesting functional significance for these genes to the organism. Due to their role in detoxification, M. tuberculosis EH's have been identified as potential drug targets. Here, we demonstrate epoxide hydrolase activity of M. thermoresistibile epoxide hydrolase A (Mth-EphA) and report its crystal structure in complex with the inhibitor 1,3-diphenylurea at 2.0 Å resolution. Mth-EphA displays high sequence similarity to its orthologue from M. tuberculosis and generally high structural similarity to α/ß-hydrolase EHs. The structure of the inhibitor bound complex reveals the geometry of the catalytic residues and the conformation of the inhibitor. Comparison to other EHs from mycobacteria allows insight into the active site plasticity with respect to substrate specificity. We speculate that mycobacterial EHs may have a narrow substrate specificity providing a potential explanation for the genetic repertoire of epoxide hydrolase genes in M. tuberculosis.

"I'm Kinda Stuck at Home With Unsupportive Parents Right Now": LGBTQ Youths' Experiences With COVID-19 and the Importance of Online Support.

PURPOSE: LGBTQ youth are a population who experience unique stressors. This study investigated their experiences with the COVID-19 pandemic via Q Chat Space-a national online chat-based support program. METHODS: Transcript data from 31 synchronous, text-based chats collected during the onset of state-based "social distancing" ordinances in Spring 2020 were analyzed. RESULTS: While encountering COVID-19-related stressors likely to be experienced by youth generally, participants' experiences were concomitantly imbued with LGBTQ-specific intrapersonal, interpersonal, and structural challenges. Difficulties included maintaining mental health, being isolated with unsupportive families, and loss of in-person identity-based socialization and support. CONCLUSIONS: Findings highlight the importance of synchronous, text-based online platforms to enable LGBTQ youth to feel safe to seek support while at home. Given the potential for long-term physical distancing, concerted efforts are required to provide necessary resources and support for LGBTQ youth during the COVID-19 pandemic.

Human oocytes harboring damaged DNA can complete meiosis I.

OBJECTIVE: To determine whether human oocytes possess a checkpoint to prevent completion of meiosis I when DNA is damaged. DESIGN: DNA damage is considered a major threat to the establishment of healthy eggs and embryos. Recent studies found that mouse oocytes with damaged DNA can resume meiosis and undergo germinal vesicle breakdown (GVBD), but then arrest in metaphase of meiosis I in a process involving spindle assembly checkpoint (SAC) signaling. Such a mechanism could help prevent the generation of metaphase II (MII) eggs with damaged DNA. Here, we compared the impact of DNA-damaging agents with nondamaged control samples in mouse and human oocytes. SETTING: University-affiliated clinic and research center. PATIENT(S): Patients undergoing ICSI cycles donated GV-stage oocytes after informed consent; 149 human oocytes were collected over 2 years (from 50 patients aged 27-44 years). INTERVENTIONS(S): Mice and human oocytes were treated with DNA-damaging drugs. MAIN OUTCOME MEASURE(S): Oocytes were monitored to evaluate GVBD and polar body extrusion (PBE), in addition to DNA damage assessment with the use of γH2AX antibodies and confocal microscopy. RESULT(S): Whereas DNA damage in mouse oocytes delays or prevents oocyte maturation, most human oocytes harboring experimentally induced DNA damage progress through meiosis I and subsequently form an MII egg, revealing the absence of a DNA damage-induced SAC response. Analysis of the resulting MII eggs revealed damaged DNA and chaotic spindle apparatus, despite the oocyte appearing morphologically normal. CONCLUSION(S): Our data indicate that experimentally induced DNA damage does not prevent PBE in human oocytes and can persist in morphologically normal looking MII eggs.

The Effect of Assisted Hatching on Live Birth Rate Following Fresh Embryo Transfer in Advanced Maternal Age.

OBJECTIVE: Assisted hatching (AH) was introduced 3 decades ago as an adjunct method to in vitro fertilization (IVF) and embryo transfer (ET) to improve embryo implantation rate. Limited data are available on the effect of AH on live birth rate (LBR) in advanced maternal age. The objective of this study is to investigate the effect of AH on LBR in women aged 40 years and older. MATERIALS AND METHODS: A retrospective study conducted at a single academic reproductive center. Women aged ≥40 years, who were undergoing their first IVF cycle were included. Laser-assisted hatching was the method used for AH and single or double embryos were transferred. Embryo transfer was performed at the cleavage or blastocyst stage. Separate analysis was performed on each ET stage. Live birth rate was the primary outcome. RESULTS: A total of 892 patients were included. Of these, 681 women underwent cleavage ET and 211 underwent blastocyst ET. The clinical pregnancy rate in the entire group was 15.3% and the LBR was 10.2%. Baseline and cycle parameters between the AH group and the control group were comparable. Assisted hatching in the cleavage stage was associated with lower clinical pregnancy rate (odds ratio [OR], 0.52; confidence interval [CI], 0.31-0.86; P = .012) and lower LBR (OR, 0.36; CI, 0.19-0.68; P = .001). Assisted hatching did not have any effect on outcomes in blastocyst ET. CONCLUSION: Assisted hatching does not improve the reproductive outcomes in advanced maternal age. Performing routine AH for the sole indication of advanced maternal age is not clinically justified.

Immature Oocyte for Fertility Preservation.

In vitro maturation (IVM) of human immature oocytes has been offered to women who are at risk of developing ovarian hyperstimulation syndrome (OHSS) caused by gonadotropin stimulation, such as PCO(S) patients or who have poor ovarian reserve. Cryopreservation of oocytes matured in vivo obtained in IVF cycles has improved after implementing the vitrification method and many successful results have been reported. Now, this procedure can be successfully offered to fertility preservation programs for patients who are in danger of losing their ovarian function due to medical or social reasons, and to oocyte donation programs. This vitrification technique has also been applied to cryopreserve oocytes obtained from IVM program. Some advantages of oocytes vitrification related with IVM are: (1) eliminating costly drugs and frequent monitoring; (2) completing treatment within 2 to 10 days (3) avoiding the use of hormones in cancer patients with hormone-sensitive tumors; and (4) retrieving oocytes at any point in menstrual cycle, even in the luteal phase. In addition, immature oocytes can also be collected from extracorporeal ovarian biopsy specimens or ovaries during caesarian section. Theoretically, there are two possible approaches for preserving immature oocytes: oocyte cryopreservation at the mature stage (after IVM) and oocyte cryopreservation at the Germinal Vesicle (GV)-stage (before IVM). Both vitrification of immature oocyte before/after IVM is not currently satisfactory. Nevertheless, many IVF centers worldwide are doing IVM oocyte cryopreservation as one of the options to preserve fertility for female cancer. Therefore, more studies are urgently required to improve IVM- and vitrification method to successfully preserve oocytes collected from cancer patients. In this review, present oocyte maturation mechanisms and recent progress of human IVM cycles will be discussed first, followed by some studies of the vitrification of human IVM oocyte.