Ex ovo omnia-why don't we know more about egg quality via imaging?

Determining egg quality is the foremost challenge in assisted reproductive technology (ART). Although extensive advances have been made in multiple areas of ART over the last 40 years, oocyte quality assessment tools have not much evolved beyond standard morphological observation. The oocyte not only delivers half of the nuclear genetic material and all of the mitochondrial DNA to an embryo but also provides complete developmental support during embryonic growth. Oocyte mitochondrial numbers far exceed those of any somatic cell, yet little work has been done to evaluate the mitochondrial bioenergetics of an oocyte. Current standard oocyte assessment in in vitro fertilization (IVF) centers include the observation of oocytes and their surrounding cell complex (cumulus cells) via stereomicroscope or inverted microscope, which is largely primitive. Additional oocyte assessments include polar body grading and polarized light meiotic spindle imaging. However, the evidence regarding the aforementioned methods of oocyte quality assessment and IVF outcomes is contradictory and non-reproducible. High-resolution microscopy techniques have also been implemented in animal and human models with promising outcomes. The current era of oocyte imaging continues to evolve with discoveries in artificial intelligence models of oocyte morphology selection albeit at a slow rate. In this review, the past, current, and future oocyte imaging techniques will be examined with the goal of drawing attention to the gap which limits our ability to assess oocytes in real time. The implications of improved oocyte imaging techniques on patients undergoing IVF will be discussed as well as the need to develop point of care oocyte assessment testing in IVF labs.

The Association of Racism and Discrimination in Disparities of Hypertensive Disorders of Pregnancy in the United States: An Analysis of PRAMS Data.

BACKGROUND: Hypertensive disorders of pregnancy are a leading cause of maternal mortality. Racial disparities in maternal outcomes such as maternal mortality in the United States (US) are well-documented, but the relationship of racism and/or discrimination with one's risk of developing a hypertension during pregnancy (HDP) is not well-studied. METHODS: Data from 17 sites that asked questions regarding experiences with racism and/or discrimination during pregnancy via the Pregnancy Risk Assessment Monitoring System (PRAMS) Phase 8 (2016-2020) was used. Logistic regression models were used compare the potential of stress induced HDP from perceived experiences of racism and/or discrimination versus the effect of systemic racism and/or discrimination (in healthcare settings and generally) on racial disparities in HDP diagnosis. RESULTS: Among 9,907 live births, 18% of participants reported they were diagnosed with hypertension during pregnancy, with non-Hispanic Black individuals having the highest rate (21.8%). Regarding experiences of racism and/or discrimination, 76.4% of participants responded "yes", with all races/ethnicities studied here except non-Hispanic White individuals responding "yes" at rates higher than 89%. Perceived experiences of racism and/or discrimination did not statistically significantly affect one's odds of being diagnosed with HDP (OR = 0.94, CI: 0.74, 1.20). The disparity in odds of having hypertension during pregnancy between Non-Hispanic Black individuals and non-Hispanic White individuals was not statistically significant when perceived experiences of racism and/or discrimination were included in the model. CONCLUSIONS: Experiences of racism and/or discrimination drive racial disparities in hypertensive disorders in pregnancy. PUBLIC HEALTH IMPLICATIONS: It is vital to eliminate racist and discriminatory practices and behaviors to reduce maternal morbidity and mortality.

Multidrug resistance transporter-1 dysfunction perturbs meiosis and Ca2+ homeostasis in oocytes.

In brief: Oocyte quality remains the most important and unsolved issue in reproduction. Our data show that multidrug resistance transporters and oocyte mitochondria are involved in determining oocyte quality in a mouse model. Abstract: Multidrug resistance transporter-1 (MDR-1) is a transmembrane ATP-dependent effluxer present in organs that transport a variety of xenobiotics and by-products. Previous findings by our group demonstrated that this transporter is also present in the oocyte mitochondrial membrane and that its mutation led to abnormal mitochondrial homeostasis. Considering the importance of these organelles in the female gamete, we assessed the impact of MDR-1 dysfunction on mouse oocyte quality, with a particular focus on the meiotic spindle organization, aneuploidies, Ca2+ homeostasis, ATP production and mtDNA mutations. Our results demonstrate that young Mdr1a mutant mice produce oocytes characterized by lower quality, with a significant delay in the germinal vesicle to germinal vesicle breakdown transition, an increased percentage of symmetric divisions, chromosome misalignments and a severely altered meiotic spindle shape compared to the wild types. Mutant oocytes exhibit 7000 more SNPs in the exomic DNA and twice the amount of mitochondrial DNA (mtDNA) SNPs compared to the wild-type ones. Ca2+ analysis revealed the inability of MDR-1 mutant oocytes to manage Ca2+ storage content and oscillations in response to several stimuli, and ATP quantification shows that mutant oocytes trend toward lower ATP levels compared to wild types. Finally, 1-year-old mutant ovaries express a lower amount of SIRT1, SIRT3, SIRT5, SIRT6 and SIRT7 compared to wild-type levels. These results together emphasize the importance of MDR-1 in mitochondrial physiology and highlight the influence of MDR-1 on oocyte quality and ovarian aging.

The DIY IVF cycle-harnessing the power of deeptech to bring ART to the masses.

The emergence of telehealth including telemedicine, at-home testing, and mobile health applications has enabled patients to self-manage their reproductive care, especially during the COVID-19 pandemic. Reproduction is rapidly changing and embracing deeptech initiatives that can improve outcomes and facilitate personalized fertility solutions in the near future. This so-called DIY IVF informed by deeptech and moderated by femtech not only holds a tremendous amount of promise, but also challenges and possible pitfalls. This review discusses the current status of deeptech and femtech for IVF care in a post-Roe v. Wade environment.

Infertility and assisted reproductive technology outcomes in Afro-Caribbean women.

OBJECTIVE: To assess the causes of infertility and artificial reproductive technology (ART) outcomes in women of African descent living in the Caribbean and Bermuda. DESIGN: Cross-sectional study composed of a questionnaire administered to providers who care for women undergoing ART in the Caribbean and Bermuda. MATERIAL AND METHODS: A questionnaire from the Deerfield Institute was adapted to meet the aims of our study with their permission. Eight infertility clinics in the Caribbean and Bermuda were identified. The primary physician at each site was contacted via email and invited to participate in the study. Questionnaires were completed via interview or electronically. Responses were collected in a REDCap database for statistical analysis. RESULTS: There were five respondents from Barbados, Bermuda (× 2), Puerto Rico, and the Bahamas. The most commonly reported etiologies of infertility among Afro-Caribbean patients were female-male factor and uterine factor. In vitro fertilization (IVF) combined with intracytoplasmic sperm injection (ICSI) is performed more often than conventional IVF. The cumulative live birth rates (LBR) after ART for those ages ≤ 34, 35-37, 38-42, and > 42 were 52%, 40%, 22%, and 12%, respectively. The cumulative live birth rate was 31.5% for total patients. The factors reported to be most important in hindering patients from cycling were coping emotionally with poor ovarian response and cost. The biggest restraints to infertility care were costs and a lack of local IVF centers on all islands. CONCLUSION: Afro-Caribbean women receiving infertility care in the Caribbean may have better ART outcomes compared to African-American women in the United States (US).

The use of technology in the sexual health education especially among minority adolescent girls in the United States.

PURPOSE OF REVIEW: The purpose of this review is to examine current sexual health education technologies for adolescents and identify gaps in knowledge. Disparities in sexual health education are prominent and mainly affect young women of color. As a result, the use of technology to reach these marginalized populations could potentially invoke change. Thus, it is crucial to determine the viability of technology as a mechanism to bridge the knowledge gap regarding sexual health for adolescents that are most at risk of sexually transmitted infections and unwanted pregnancies. RECENT FINDINGS: With a lack of standardized, evidenced-based sexual health education programs in the United States, the future of comprehensive sexual health education is moving toward smartphone apps. Many sexual health technologies exist that target adolescents, most of which have been proven to demonstrate positive effects. Use of mobile apps, especially for vulnerable populations, can be more effective because of privacy and widespread dissemination. SUMMARY: Ultimately, more research needs to be conducted to determine the most effective content for these sexual health apps. Additionally, more research should be conducted on effective sexual health apps for marginalized populations to determine whether technology is a viable solution.

The Role of T Cells in Ovarian Physiology and Infertility.

Infertility affects one in six couples worldwide, with more than 48 million couples affected internationally. The prevalence of infertility is increasing which is thought to be attributed to delayed child-bearing due to socioeconomic factors. Since women are more prone to autoimmune diseases, we sought to describe the correlation between ovarian-mediated infertility and autoimmunity, and more specifically, the role of T cells in infertility. T cells prevent autoimmune diseases and allow maternal immune tolerance of the semi-allogeneic fetus during pregnancy. However, the role of T cells in ovarian physiology has yet to be fully understood.

Factors associated with disparate outcomes among Black women undergoing in vitro fertilization.

Objective: To determine if Black women have worse in vitro fertilization (IVF) outcomes than women of other races/ethnicities, and to establish which factors are associated with the IVF outcomes of Black women. Design: Retrospective cohort study. Setting: Not applicable. Patients: All patients undergoing IVF. Interventions: Not applicable. Main Outcome Measures: Spontaneous abortion rate, clinical pregnancy rate, and live birth rate. Results: A total of 71,389 patient cycles were analyzed. Of the 40,545 patients who were included, 6.4% of patients were Black, 62% were White, 7.3% were Hispanic/Latino, and 15% were Asian. After IVF, Black women had significantly more miscarriages than White but not Hispanic or Asian patients (8.0% Black vs. 6.9% White, 7.4% Hispanic, and 7.5% Asian). Clinical pregnancy rates were significantly lower for Black women compared with all other races (45% Black vs. 52% White, 52% Hispanic, and 53% Asian). The odds ratio (OR) of live birth from all cycles were 30% less than that for White women (OR, 1.00 Black vs. 1.43 White) and 22% less than that for Hispanic women (OR, 1.00 Black vs. 1.29 Hispanic). This statistically significant difference in the live birth rate persisted even after adjusting for patient characteristics (OR, 1.00 Black vs. 1.32 White, 1.23 Hispanic, and 1.18 Asian). Conclusions: Black women have worse IVF outcomes than women of all other racial backgrounds undergoing IVF. The factors associated with the disparate outcomes of Black women undergoing IVF outcomes include older age starting IVF, higher body mass index, tubal factor infertility, and diabetes.

MDR-1 function protects oocyte mitochondria against the transgenerational effects of nitrogen mustard exposure.

Oocytes are vulnerable to alkylating agents like nitrogen mustard (NM), which can cause mitochondrial dysfunction associated with increased oxidative stress. Because mitochondria are maternally inherited, NM exposure affects oocyte mitochondrial physiology and compromises future progeny. Multidrug resistance transporters (MDRs) are transmembrane proteins that efflux such cytotoxic substances; MDR-1 is expressed in oocyte plasma and mitochondrial membranes and protects against oxidative stress. Our objective was to investigate how loss of MDR-1 can modulate oocyte response to NM transgenerationally. Wild Type (WT) and Mdr1a mutant female mice were injected intraperitoneally with sterile saline (control) or 0.1 mg/kg NM. 48 h post-injection, females were either sacrificed for F0 studies or mated with control males to yield F1 pups. After weaning, F1 females were sacrificed or mated to yield F2 pups. Germinal vesicle oocytes were assessed for mitochondrial membrane potential and reactive oxygen species (ROS) levels. NM exposed oocytes of both genotypes exhibited significantly higher ROS than controls in F0 and F1. NM F2 oocytes of neither genotype exhibited significantly higher ROS, though variation in Mdr1a mutants led to an upward trend. NM oocytes of both genotypes exhibited significantly disrupted mitochondrial membrane potential in F0. WT regained normalcy by F1 whereas Mdr1a mutants were unable to by F2. Our data suggest that Mdr1a mutants exhibit transgenerational mitochondrial dysfunction following toxic challenge that persists, implying that MDR-1 protects against toxicant-induced mitochondrial stress. Women without functional MDR-1 exposed to environmental toxicants could therefore be at risk for passing on compromised mitochondria to future offspring.

Infertility knowledge and treatment beliefs among African American women in an urban community.

BACKGROUND: To assess infertility knowledge and treatment beliefs among African American women in an urban community in Atlanta, Georgia. METHODS: This was a cross sectional study at a safety net hospital. A convenience sample of a total of 158 women receiving outpatient obstetrical or gynecologic care from March-April 2017 were recruited. Infertility knowledge and treatment beliefs were assessed using a previously applied and field-tested survey from the International Fertility Decision Making Study. RESULTS: The mean infertility knowledge score was 38.15% for total subjects. Those with a higher level of education (p < 0.0001) and those with paid employment (p = 0.01) had a significantly higher level of infertility knowledge. Those who had a history of infertility therapy were significantly more likely to agree with negative treatment beliefs (p = 0.01). There was no significant difference in infertility knowledge or treatment beliefs based on age, sexuality, parity or being pregnant at the time of survey completion. CONCLUSIONS: African American women in our urban clinic setting seem to have a limited level of knowledge pertaining to infertility. Further research is needed to understand how differences in knowledge and beliefs translate into infertility care decision-making and future childbearing.

Dysfunctional MDR-1 disrupts mitochondrial homeostasis in the oocyte and ovary.

Multidrug resistance transporters (MDRs) are best known for their pathological role in neoplastic evasion of chemotherapeutics and antibiotics. Here we show that MDR-1 is present in the oocyte mitochondrial membrane, and it protects the female gamete from oxidative stress. Female mdr1a mutant mice have no significant difference in ovarian follicular counts and stages, nor in reproductively functioning hormone levels, yet these mice are significantly more vulnerable to gonadotoxic chemotherapy, have chronically elevated reactive oxygen species in immature germinal vesicle oocytes, exhibit a significant over-accumulation of metabolites involved in the tricarboxylic acid cycle (TCA), and have abnormal mitochondrial membrane potential. The mdr1a mutant ovaries have a dramatically different transcriptomic profile with upregulation of genes involved in metabolism. Our findings indicate that functionality of MDR-1 reveals a critical intersection of metabolite regulation, oxidative stress, and mitochondrial dysfunction that has direct implications for human infertility, premature reproductive aging due to oxidative stress, and gonadoprotection.

Ovarian hormones modulate multidrug resistance transporters in the ovary.

BACKGROUND: Multidrug resistance transporters (MDRs) are transmembrane proteins that efflux metabolites and xenobiotics. They are highly conserved in sequence and function in bacteria and eukaryotes and play important roles in cellular homeostasis, as well as in avoidance of antibiotics and cancer therapies. Recent evidence also documents a critical role in reproductive health and in protecting the ovary from environmental toxicant effects. The most well understood MDRs are MDR-1 (P-glycoprotein (P-gp) also known as ABCB1) and BCRP (breast cancer resistance protein) and are both expressed in the ovary. We have previously shown that MDR-1 mRNA steady state expression changes throughout the murine estrous cycle, but expression appears to increase in association with the surge in estradiol during proestrus. METHODS: Here we test the model that MDR-1 and BCRP are regulated by estrogen, the major hormonal product of the ovary. This was performed by administering 6-week-old female mice either sesame oil (vehicle control) or oral ethinyl estradiol at 1 µg, 10 µg, and 100 µg or PROGESTERONE at 0.25mg, 0.5 mg or 1 mg or a combination of both for 5 days. The mice were then sacrificed, and the ovaries were removed and cleaned. Ovaries were used for qPCR, immunoblotting, and immnunolabeling. RESULTS: We found that oral ethinyl estradiol did not influence the steady state mRNA of MDR-1 or BCRP. Remarkably, the effect on mRNA levels neither increased or decreased in abundance upon estrogen exposures. Conversely, we observed less MDR-1 protein expression in the groups treated with 1 µg and 10 µg, but not 100 µg of ethinyl estradiol compared to controls. MDR-1 and BCRP are both expressed in pre-ovulatory follicles. When we tested progesterone, we found that MDR-1 mRNA increased at the dosages of 0.25 mg and 0.5 mg, but protein expression levels were not statistically significant. Combined oral ethinyl estradiol and progesterone significantly lowered both MDR-1 mRNA and protein. CONCLUSIONS: Progesterone appears to influence MDR-1 transcript levels, or steady state levels. This could have implications for better understanding how MDR-1 can be modulated during times of toxic exposure. Understanding the normal physiology of MDR-1 in the ovary will expand the current knowledge in cancer biology and reproduction.

Nitrogen mustard exposure perturbs oocyte mitochondrial physiology and alters reproductive outcomes.

Nitrogen mustard (NM) is an alkylating chemical warfare agent, and its derivatives are used in chemotherapy. Alkylating agents can cause mitochondrial damage, so exposed females may transmit damaged genomes to their children, since mitochondria are maternally inherited and oocytes are not thought to undergo mitophagy (Boudoures et al. [1]). The objective of this study is to investigate NM's effects on oocyte mitochondria to understand risks facing female soldiers, cancer patients, and their children. Mice were injected intraperitoneally with NM, monitored for reproductive outcomes, and ovaries and oocytes were isolated for analysis. Escalating doses of NM increased oxidative stress in parental and F1 generation oocytes, suggesting that mitochondrial damage by NM is enhanced by mitochondrial superoxide. NM-treated ovaries in vitro exhibited smaller mitochondrial volume, more electron-dense and multivesicular structures, and lower birth weight litters. These results demonstrate that females must be protected from alkylating agents for their health, and the health of their offspring.

Multidrug resistance transporter-1 and breast cancer resistance protein protect against ovarian toxicity, and are essential in ovarian physiology.

Ovarian protection from chemotoxicity is essential for reproductive health. Our objective is to determine the role of ATP-dependent, Multidrug Resistance Transporters (MDRs) in this protection. Previously we identified MDR-dependent cytoprotection from cyclophosphamide in mouse and human oocytes by use of MDR inhibitors. Here we use genetic deletions in MDR1a/b/BCRP of mice to test MDR function in ovarian somatic cells and find that mdr1a/b/bcrp-/- mice had significantly increased sensitivity to cyclophosphamide. Further, estrus cyclicity and follicle distribution in mdr1a/b/bcrp-/- mice also differed from age-matched wildtype ovaries. We found that MDR gene activity cycles through estrus and that MDR-1b cyclicity correlated with 17ß-estradiol surges. We also examined the metabolite composition of the ovary and learned that the mdr1a/b/bcrp-/- mice have increased accumulation of metabolites indicative of oxidative stress and inflammation. We conclude that MDRs are essential to ovarian protection from chemotoxicity and may have an important physiological role in the ovary.

Girl Talk: A Smartphone Application to Teach Sexual Health Education to Adolescent Girls.

STUDY OBJECTIVE: Produce Girl Talk, a free smartphone application containing comprehensive sexual health information, and determine the application's desirability and appeal among teenage girls. DESIGN, SETTING, PARTICIPANTS, AND INTERVENTIONS: Thirty-nine girls ages 12 to 17 years from Rhode Island participated in a 2-phase prospective study. In phase I, 22 girls assessed a sexual health questionnaire in focus groups. In phase II, 17 girls with iPhones used Girl Talk for 2 weeks and answered the revised sexual health questionnaire and interview questions before and after use. MAIN OUTCOME MEASURES: Participants' responses to the sexual health questionnaire, interviews, and time viewing the application were used to determine feasibility and desirability of Girl Talk. RESULTS: Girl Talk was used on average for 48 minutes during participants' free time on weekends for 10- to 15-minute intervals. Reported usefulness of Girl Talk as a sexual health application from baseline (6 participants) to follow-up (16 participants) increased significantly (35.3% vs 94.1%; P < .001). Knowledge improved most in topics related to anatomy and physiology (70.5% to 74.7% out of 7 questions), sexuality and relationships (76.5% to 80.0% out of 10 questions), and STI prevention (75.6% to 79.0% out of 7 questions). Most phase II participants (13 out of 17, or 76.5%) were exposed to sexual health education before using Girl Talk, but 16 out of 17 participants (94.1%) stated that the application provided new and/or more detailed information than health classes. CONCLUSION: Girl Talk can potentially connect teenage girls to more information about sexual health vs traditional methods, and participants recommended the application as a valuable resource to learn about comprehensive sexual health.

Simple perfusion apparatus for manipulation, tracking, and study of oocytes and embryos.

OBJECTIVE: To develop and implement a device and protocol for oocyte analysis at a single cell level. The device must be capable of high resolution imaging, temperature control, perfusion of media, drugs, sperm, and immunolabeling reagents all at defined flow rates. Each oocyte and resultant embryo must remain spatially separated and defined. DESIGN: Experimental laboratory study. SETTING: University and academic center for reproductive medicine. PATIENT(S)/ANIMAL(S): Women with eggs retrieved for intracytoplasmic sperm injection (ICSI) cycles, adult female FVBN and B6C3F1 mouse strains, sea stars. INTERVENTION(S): Real-time, longitudinal imaging of oocytes after fluorescent labeling, insemination, and viability tests. MAIN OUTCOME MEASURE(S): Cell and embryo viability, immunolabeling efficiency, live cell endocytosis quantification, precise metrics of fertilization, and embryonic development. RESULT(S): Single oocytes were longitudinally imaged after significant changes in media, markers, endocytosis quantification, and development, all with supreme control by microfluidics. Cells remained viable, enclosed, and separate for precision measurements, repeatability, and imaging. CONCLUSION(S): We engineered a simple device to load, visualize, experiment, and effectively record individual oocytes and embryos without loss of cells. Prolonged incubation capabilities provide longitudinal studies without need for transfer and potential loss of cells. This simple perfusion apparatus provides for careful, precise, and flexible handling of precious samples facilitating clinical IVF approaches.

Multidrug-resistant transport activity protects oocytes from chemotherapeutic agents and changes during oocyte maturation.

OBJECTIVE: To determine the multidrug-resistant transporter (MDR) activity in oocytes and their potential role in oocyte susceptibility to chemotherapy. DESIGN: Experimental laboratory study. SETTING: University and academic center for reproductive medicine. SUBJECT(S): Women with eggs retrieved for intracytoplasmic sperm injection cycles and adult female FVBN and B6C3F1 mouse strains. INTERVENTION(S): Inhibition of MDR activity in oocytes. MAIN OUTCOME MEASURE(S): Efflux activity of MDRs with the use of quantitative fluorescent dye efflux, and oocyte cell death when exposed to chemotherapy. RESULT(S): Oocytes effluxed fluorescent reporters, and this activity was significantly reduced in the presence of the MDR inhibitor PSC 833. Geminal vesicle oocytes were more efficient at efflux than metaphase 2 oocytes. Human oocytes exposed to cyclophosphamide and PSC 833 showed cell death with the use of two different viability assays compared with control samples and those exposed to cyclophosphamide alone. Immunoblots detected MDR-1 in all oocytes, with the greatest accumulation in the geminal vesicle stage. CONCLUSION(S): Oocytes have a vast repertoire of active MDRs. The implications of this study are that these protective mechanisms are important during oogenesis and that these activities change with maturation, increasing susceptibility to toxicants. Future directions may exploit the up-regulation of these transporters during gonadotoxic therapy.

Subclinical elevations of thyroid-stimulating hormone and assisted reproductive technology outcomes.

The prevalence of moderately elevated TSH levels consistent with subclinical hypothyroidism (2.5-4.0 µIU/mL) was 23% in a cohort of 1,231 women pursuing assisted reproductive technologies. Preconception elevated levels of TSH were associated with diminished ovarian reserve but were not associated with adverse assisted reproductive technology or pregnancy outcomes.