Survey of colleges and schools of pharmacy to determine restrictions for teaching, research, and advocacy related to contraception.

INTRODUCTION: The pharmacist's role in reproductive health is evolving. Since 46 states allow providers to refuse to provide reproductive health services, it is important to consider whether learning is impacted by institution restrictions on contraception teaching, advocacy, and research. METHODS: An electronic survey was emailed to deans of all pharmacy schools on the American Association of Colleges of Pharmacy Institutional Membership list with a request to share with faculty teaching women's health content within their curriculum. The survey collected information about contraception teaching, research, and advocacy. RESULTS: Of 145 schools contacted, 39 (27%) provided complete responses. Of these, 22 (56%) were public, not religiously-affiliated, seven (18%) were private, not religiously-affiliated, six (15%) were private, currently religiously-affiliated, and four were (10%) private, historically religiously-affiliated. All respondents taught hormonal contraception in the required curriculum and 15 (39%) taught miscarriage management/abortifacients. None reported restrictions on contraception teaching or research. One respondent cited an advocacy restriction for contraception methods due to violation of the school's beliefs, and another cited an advocacy restriction for miscarriage management/abortifacients. Respondents noted students expressed ethical questions/concerns about refusing to dispense contraception (59%), dispensing certain contraceptives (54%), dispensing to minors (46%), and dispensing all contraceptives (21%). Additionally, respondents reported pharmacists/faculty expressed ethical questions/concerns about refusing to dispense contraception (31%), dispensing to minors (21%), dispensing certain contraceptives (15%), and all contraceptives (13%). CONCLUSIONS: Overall, respondents reported no restrictions in contraception teaching and scholarship and minimal advocacy restrictions. Faculty should consider ethical questions/concerns from students, faculty, and pharmacists when teaching this material.

Using the Theory of Planned Behavior to assess community pharmacists' intention to prescribe using the Formulary and Protocol Compendia.

BACKGROUND: Oregon pharmacists can autonomously prescribe hormonal contraception, naloxone, and various medications and devices from a Formulary and Protocol Compendia (FPC). Prescribing using the FPC has seen limited uptake. OBJECTIVES: (1) Assess Oregon community pharmacists' intention to prescribe using the FPC; (2) Determine the impact that attitudes, subjective norms (SN), perceived behavioral control (PBC), perceived obligation, and past prescribing behavior have on pharmacists' intention to prescribe; (3) Examine the relationship between beliefs and pharmacists' attitudes, subjective norms, and perceived behavioral control. METHODS: This study was conducted as a cross sectional survey, which was developed based on the results of a previously conducted research project. A modified version of the Theory of Planned Behavior (TPB) was the study framework. All Oregon community pharmacists with a public email address were invited to participate. The questionnaire collected information on TPB constructs as well as demographic and practice information. TPB constructs were measured directly (intention, attitudes, SN, PBC, perceived obligation, and past prescribing behavior) and indirectly (attitudes, SN, and PBC). Descriptive statistics were used for all items. Multiple linear regression was used to assess Objectives 2 and 3. RESULTS: There were 175 useable responses included in the analysis of the estimated 1015 community pharmacists that received the survey invitation (17%). Respondents had 16.5 ± 13.3 years in practice, 60% held a PharmD degree, and 69% had prescribed in some manner before. Mean intention to prescribe using the FPC was 5.0 ± 1.5 (7-point scale with 7 indicating higher intention). Attitudes, SN, PBC, and PO were significant predictors of intention to prescribe, while past prescribing behavior was not (Adj R2 = 0.741, p < .0001). Attitudes were explained by beliefs about increasing patient access (p = .0179). PBC was explained by beliefs about having policies and procedures in place (p = .004) and feeling comfortable prescribing (p = .008). CONCLUSIONS: Oregon community pharmacists have a positive intention to prescribe using the FPC, but actual uptake remains low. Efforts to increase uptake should focus on the beliefs that contribute most strongly to pharmacist intention.

Pharmacists' intention to prescribe under new legislation.

BACKGROUND: In 2017, Oregon passed legislation that authorized pharmacists to autonomously prescribe specified medications or devices that were included on the Formulary and Protocol Compendia (FPC). Factors that impact pharmacists' intention to prescribe from the FPC are not currently known. OBJECTIVES: Identify factors that influence pharmacists' intention to prescribe. METHODS: The Theory of Planned Behavior (TPB) served as the framework. Three focus groups were conducted with Oregon pharmacists between May and June 2019 to elicit salient beliefs about prescribing related to TPB constructs, including attitudes, subjective norms, perceived behavioral control, and perceived obligation. Two investigators conducted a qualitative content analysis and reached agreement on names of categories during consensus meetings. A codebook was created after analysis of the first focus group and used to guide the remaining analysis. Participants reported background information and awareness of prescribing rules via a questionnaire; descriptive statistics were used to report background information and the prescribing awareness summary score. RESULTS: Thirteen participants had 14.1 ± 10.8 years of pharmacist experience. Most earned a Doctor of Pharmacy degree (84.6%) and worked in a community pharmacy setting (61.5%). The mean score on the awareness assessment was 6.3 ± 0.9 (7 = highest awareness). The majority (76.9%) had previous prescribing experience. Attitudes were found to be shaped by behavioral beliefs related to the impact of pharmacist prescribing on patient-centered care and on practice transformation. Normative beliefs were primarily driven by outside groups past experiences with pharmacists and pharmacies. Control beliefs included the following categories: operational readiness; the relationships between comfort, competence, and confidence; and Board of Pharmacy requirements. CONCLUSIONS: TPB was useful for exploring pharmacists' beliefs related to their intention to prescribe using the FPC. Determining the relative importance of these factors in a broader population will enable stakeholders to develop interventions to improve uptake of prescribing via the FPC.

Association of pharmacist prescription and 12-month contraceptive continuation rates.

BACKGROUND: States have passed legislation to expand the scope of pharmacists to directly prescribe contraception. It is thought that pharmacist prescription of contraception may promote correct and consistent use of contraception by reducing barriers to access. However, it is not known how this may impact ongoing contraceptive use. OBJECTIVE: This study aimed to determine whether 12-month rates of continuation of an effective form of contraception or perfect use of contraception differ by prescribing provider (pharmacist or clinician). STUDY DESIGN: We conducted a 1-year prospective cohort study of 388 women seeking contraception in 139 pharmacies across 4 states (California, Colorado, Hawaii, and Oregon). Our study was powered to detect a 10% difference in 12-month continuation of an effective form of contraception. We clarified women's pregnancy intention at baseline and subsequent follow-ups. Women received a prescription directly from a pharmacist (n=149) or were filling a prescription from a clinician, our comparison group (n=239). We used multivariable logistic regression to measure the association between pharmacist prescriber and use of any effective contraceptive method or perfect use at 12 months. Model covariates included age, race, education, side effects experienced, payor, and contraceptive supply dispensed at baseline. RESULTS: Of the study cohort, 88% (n=340) completed 12 months of follow-up. Among women not planning to become pregnant, 7 women in the clinic-prescribed group vs 1 woman in the pharmacy-prescribed group (3.4% vs 0.8%; P>.05) reported a positive pregnancy test during the study period. The majority of the cohort was continuing to use an effective method of contraception at 12 months (clinician 89.3% vs pharmacist 90.4%; P=.86). Among women receiving a prescription from a clinician, 53.9% reported perfect use (no missed days) at 12 months, compared with 47% of the pharmacist-prescribed group (P=.69). Pharmacist prescriber type was not associated with continuation of an effective contraceptive method at 12 months (adjusted odds ratio, 0.70; confidence interval, 0.28-1.71) or with perfect use of contraception (adjusted odds ratio, 0.87; confidence interval, 0.51-1.48), controlling for other woman-level characteristics. CONCLUSION: We found no difference in use of any effective contraception, perfect use, or switching at 12 months among those who received their baseline prescription from a pharmacist vs a clinician. This study is limited by not examining information on safety outcomes.

Medical contraindications to combined hormonal contraceptive use among women using methods prescribed by a pharmacist.

OBJECTIVE: To determine whether pharmacist prescription of combined hormonal contraception is associated with inappropriate prescription to women with medical contraindications. STUDY DESIGN: We conducted a retrosopective cohort study of all short-acting, hormonal contraceptive users (pill, patch, ring, injectable) in Oregon's All Payer All Claims database from January 1, 2016 to December 31, 2018. Our primary outcome was the proportion of women receiving a combined hormonal method who had a Medical Eligibility Category (MEC) 3 or 4 condition. We identified potential contraindications using International Classification of Disease codes. We conducted descriptive analyses of contraindication prevalence and prescription error rate by prescriber type. We used a multivariable logistic regression model to test the association between pharmacist prescriber and population characteristics. RESULTS: Our study sample consisted of 439,240 contraceptive users, of which 3782 (0.86%) received their prescriptions from a pharmacist. Women aged 25 to 29 were more likely than women over age 35 to receive contraception from a pharmacist (adjusted odds ratio (aOR) 2.74, 95% confidence interval [CI] 2.44-3.08). Pharmacist prescriptions were slightly less likely in rural areas (aOR 0.78, 95% CI 0.69-0.89) and among women on Medicaid, relative to those with commercial insurance (aOR 0.21, 95% CI 0.19-0.24). Among women given contraception in a clinical setting, 4.25% had evidence of an MEC 3 or 4 contraindication, compared to 0.9% for women seen by a pharmacist. Rates of prescribing a combined method to women with a potential contraindication were not meaningfully different by prescriber type (2.16% for clinicians vs 0.74% for pharmacists). CONCLUSION: Rates of contraceptive prescribing with a contraindication were relatively low and did not differ between clinicians and pharmacists. IMPLICATIONS: Pharmacists can safely screen for medical contraindications to combined hormonal contraception.

Association of Pharmacist Prescription With Dispensed Duration of Hormonal Contraception.

Importance: Since 2016, 11 states have expanded the scope of pharmacists to include direct prescription of hormonal contraception. Dispensing greater than 1 month's supply is associated with improved contraceptive continuation rates and fewer breaks in coverage. Scant data exist on the practice of pharmacist prescription of contraception and its outcomes compared with traditional, clinic-based prescriptions. Objective: To compare the amount of hormonal contraceptive supply dispensed between pharmacists and clinic-based prescriptions. Prescribing patterns were assessed by describing prescribing practices for women with contraindications to combined hormonal contraception. Characteristics of women seeking hormonal contraception directly from pharmacists were also described. Design, Setting, and Participants: This cohort study surveyed women aged 18 to 50 years who presented to pharmacies in California, Colorado, Hawaii, and Oregon for hormonal contraception prescribed by a clinician or a pharmacist between January 30 and November 1, 2019. Exposures: Pharmacist or clinic-based prescription of contraception. Main Outcomes and Measures: Months of contraceptive supply dispensed. Results: Four hundred ten women (mean [SD] age, 27.1 [7.7] years) were recruited who obtained contraception directly from a pharmacist (n = 144) or by traditional clinician prescription (n = 266). Women obtaining contraception from a pharmacist were significantly younger (82 [56.9%] vs 115 [43.2%] participants aged 18-24 years; P = .03), had less education (38 [26.4%] vs 100 [37.6%] with a bachelor degree; P = .002), and were more likely to be uninsured (16 [11.1%] vs 8 [3.0%] participants; P = .001) compared with women with a prescription from a clinician. Pharmacists were significantly more likely to prescribe a 6-month or greater supply of contraceptives than clinicians (6.9% vs 1.5%, P < .001) and significantly less likely to only prescribe a 1-month supply (42 [29.2%] vs 118 [44.4%] prescriptions; P < .001). Controlling for all covariates, women seen by pharmacists had higher odds of receipt of a 6-month or greater supply of contraceptives compared with those seen by clinicians (odds ratio = 3.55; 95% CI, 1.88-6.70). Pharmacists were as likely as clinicians to prescribe a progestin-only method to women with a potential contraindication to estrogen (n = 60 women; 8 [20.0%] vs 6 [30.0%], P = .52). Conclusions and Relevance: These findings suggest that pharmacist prescription of contraception may be associated with improved contraceptive continuation by preventing breaks in coverage through the provision of a greater supply of medication. Efforts are needed to educate prescribing providers on the importance of dispensing 6 months or greater contraceptive supply.

Preparing Maize Synaptonemal Complex Spreads and Sequential Immunofluorescence and Fluorescence In Situ Hybridization.

Immunofluorescence and fluorescence in situ hybridization (FISH) can be used to locate specific proteins and DNA sequences, respectively, in chromosomes by light microscopy. Here we describe sequential use of these techniques on spreads of maize synaptonemal complexes (SCs) to determine whether crossing over can occur in knob heterochromatin. We used antibodies to AFD1, an SC protein, and MLH1, a class I (interference-sensitive) crossover protein found in most recombination nodules (RNs) to identify crossovers (COs) along SCs. Next, we used FISH to localize a 180 bp knob-specific tandem repeat. Combining immunofluorescence and FISH images of the same SC spreads showed that heterochromatic knobs do not prohibit class I COs. This technique is broadly applicable to investigations of plant prophase I chromosomes where meiotic recombination takes place.

Pharmacist Provision of Hormonal Contraception in the Oregon Medicaid Population.

OBJECTIVE: To describe early utilization of pharmacist prescription of contraception in Oregon's Medicaid program. METHODS: Using Oregon Medicaid claims data, we conducted a retrospective analysis and quantified overall and monthly trends in pharmacist-prescribed contraceptives from January 1, 2016, to December 31, 2017. Our population was restricted to patients obtaining a new prescription for oral and transdermal methods and who had continuous Medicaid coverage during the study period. We summarized demographic and utilization characteristics, including whether patients were continuing or switching methods or initiating contraception. New prescriptions were those written to patients who did not have one for hormonal contraception in the prior 30 days. To assess program safety, we examined rates of prescriptions to patients with medical contraindications to contraceptive use. RESULTS: Among the 3,614 patients receiving a new prescription for oral or transdermal contraceptives in the Oregon Medicaid program from all health care providers, 367 (10%) received their prescription from a pharmacist. Five months after implementation, pharmacists filled an average of 61 prescriptions per month as the prescriber. Most claims originated from retail chain pharmacies (94%) in urban locations (71%). The majority of patients who were prescribed contraception by pharmacists (73.8%) had no history of contraceptive prescriptions in the preceding 30 days (n=252). Ages ranged from 13 to 49 years, fewer patients lived in a rural location (35.7%), most received a combined hormonal pill (90.5%), and the average day's supply dispensed was 65 (range of 21-364 days). Fewer than 5% (12) of patients had a diagnostic code indicating a possible contraindicating comorbidity. CONCLUSION: Among Medicaid enrollees, we found that 10% of all new oral and transdermal contraceptive prescriptions were written by pharmacists.

Association of Pharmacist Prescription of Hormonal Contraception With Unintended Pregnancies and Medicaid Costs.

OBJECTIVE: To estimate unintended pregnancies averted and the cost effectiveness of pharmacist prescription of hormonal contraception. METHODS: A decision-analytic model was developed to determine the cost effectiveness of expanding the scope of pharmacists to prescribe hormonal contraception compared with the standard of care and contraceptive access in clinics. Our perspective was that of the payor, Oregon Medicaid. Our primary outcome was unintended pregnancies averted. Secondary outcomes included: costs and quality-adjusted life years (QALYs). Model inputs were obtained from an analysis of Medicaid claims for the first 24 months after policy implementation in Oregon, and the literature. Univariate and bivariate sensitivity analyses, as well as a Monte Carlo simulation, were performed. RESULTS: Among Oregon's Medicaid population at risk for unintended pregnancy, the policy expanding the scope of pharmacists to prescribe hormonal contraception averted an estimated 51 unintended pregnancies and saved $1.6 million dollars. Quality of life was also improved, with 158 QALYs gained per 198,000 women. Sensitivity analysis demonstrated that the model was most sensitive to the effect on contraceptive continuation rates. If contraceptive continuation rates among women receiving care from a pharmacist are 10% less than among clinicians, than pharmacist prescription of hormonal contraception will not avert unintended pregnancies. CONCLUSION: Pharmacist prescription of hormonal contraception averts unintended pregnancies and is cost effective. Full implementation of the policy is needed for maximum benefits. Prospective data on the effect of the policy on contraceptive continuation rates are needed.

Development of a pharmacist-provided contraceptive service following passage of Oregon House Bill 2879.

OBJECTIVES: To describe the development and implementation of a pharmacist-provided contraceptive service in a community pharmacy to increase access to hormonal contraceptives and improve quality measure performance following passage of Oregon House Bill 2879. SETTING: Community pharmacy embedded within a federally qualified health center (FQHC). PRACTICE DESCRIPTION: Not applicable. PRACTICE INNOVATION: Women 18 to 50 years of age without evidence of an effective contraceptive method in the electronic medical record were administratively identified. Women were then contacted by pharmacy staff and educated about the opportunity to receive hormonal contraceptives directly from the pharmacy. Women were seen for contraceptive services through scheduled and walk-in appointments. EVALUATION: Not applicable. RESULTS: From November 1, 2017, to March 31, 2018, 23 women were seen for contraceptive services through the community pharmacy. They averaged 27.2 ± 8.6 years of age and all were Hispanic. The pharmacist prescribed hormonal contraceptives to 78.3% of the women (n = 17). Six women were referred to their primary care provider because pregnancy could not be ruled out (n = 5) or the patient requested a method not within the pharmacists' prescriptive authority (n = 1). No patients were referred to their primary care provider for elevated blood pressure readings, contraindicated disease states, or drug interactions. CONCLUSION: A pharmacist-provided contraceptive service was successfully implemented at a community pharmacy within an FQHC. This service was mostly used by young Hispanic women with unmet contraceptive needs. The expanded access to hormonal contraceptives provided by this service may help the health center to achieve quality measures related to contraceptive use and decrease unintended pregnancies in the future.

Pharmacists expand access to reproductive heaLthcare: PEARL study protocol.

BACKGROUND: In 2016, Oregon became the first of eight states to allow pharmacists to directly prescribe hormonal contraception (HC), including the pill, patch, or ring, without a clinic visit. In the two years following this policy change, the majority of ZIP codes across the state of Oregon had a pharmacist certified to prescribe HC. METHODS: We will utilize complementary methodologies to evaluate the effect of this policy change on convenient access to contraception (cost, supply dispensed), safety, contraceptive continuation and unintended pregnancy rates. We will conduct a prospective clinical cohort study to directly measure the impact of provider type on contraceptive continuation and to understand who is accessing hormonal contraception directly from pharmacists. We will concurrently conduct a retrospective analysis using medical claims data to evaluate the state-level effect of the policy. We will examine contraceptive continuation rates, incident pregnancy, and safety measures. The combination of these methodologies allows us to examine key woman-level factors, such as pregnancy intention and usual place of care, while also estimating the impact of the pharmacist prescription policy at the state level. DISCUSSION: Pharmacist prescription of HC is emerging nationally as a strategy to reduce unintended pregnancy. This study will provide data on the effect of this practice on convenient access to care, contraceptive safety and continuation rates.

Pharmacists' experience with prescribing hormonal contraception in Oregon.

OBJECTIVES: To describe Oregon pharmacy practices in provision of hormonal contraception (HC) and evaluate if pharmacists' motivation to prescribe changed after 6 and 12 months of experience. METHODS: Pharmacists practicing in Oregon who underwent the mandatory HC training were eligible to take this survey. The survey was launched 6 months after policy implementation; pharmacists who responded to the initial survey were eligible to be queried again at 12 months. Survey responses were anonymous, but pharmacists received a unique identifier so that responses could be linked between the 2 surveys. The survey consisted of pharmacy and pharmacist demographics and questions exploring attitudes toward prescribing HC and prescribing practices (e.g., cost, time needed for visit) and volume estimates. Descriptive statistics were used to analyze responses as well as the differences in key outcomes between 6 and 12 months. RESULTS: A total of 732 pharmacists were eligible to take the survey; 121 pharmacists responded to the 6-month survey (16.5% response rate), and 62 completed the 12-month survey (52% response rate). A large increase in the distribution of pharmacists prescribing HC by zip code occurred between 6 and 12 months: 19% and 63%, respectively. At both 6 and 12 months, almost one-half of all pharmacists were billing insurance for the visit, and the average visit took less than 30 minutes. The top 3 motivators for providing HC did not change over time and included increasing access, reducing unintended pregnancy, and increasing pharmacist scope of practice. CONCLUSION: One year after program implementation, pharmacist prescribing of HC continues to increase and is distributed widely across the state. A geographically diverse sample of Oregon pharmacists began prescribing of hormonal contraception within 12 months of Oregon's implementation of the new pharmacist provision policy. Their experience and the success of the program provide a roadmap for pharmacist participation to increase access to hormonal contraception.

Encouraging comprehensive and consistent pharmacist education for provision of contraception.

OBJECTIVES: To initiate a call to action for community pharmacists and key stakeholders to encourage comprehensive and consistent education and certification for contraception services, especially in states where laws have been enacted for pharmacist prescribing of hormonal contraceptives. DATE SOURCES: Websites for several boards of pharmacy that have implemented pharmacist training for contraceptive prescribing. SUMMARY: From the authors' perspective of helping to implement laws that allow pharmacist prescribing of contraception in Oregon and Colorado, lessons learned have shown that it is better to have 1 consistent resource for pharmacist certification for the following reasons: 1) Boards of pharmacy are able to ensure patient safety because all pharmacists are providing the same level of care to every patient; 2) retail chain pharmacies and pharmacy managers are assured that all their pharmacists, regardless of state, are trained in a similar and appropriate manner; and 3) pharmacists can be reimbursed through medical insurance for the patient encounter because payers are able to identify and credential pharmacists who pass an approved and accredited certification program. CONCLUSION: New laws allowing pharmacists to prescribe contraception are expanding to other states, and the implementation of these laws provides an important increase in pharmacists' scope of practice. This exciting new prospect allows the pharmacy community of each state an opportunity to coordinate and learn from each other on best practices for implementation. Having a consistent training program was identified as being one key aspect of successful implementation.

Sequential Immunofluorescent Light Microscopy and Electron Microscopy of Recombination Nodules During Meiotic Prophase I.

Immunolocalization using either fluorescence for light microscopy (LM) or gold particles for electron microscopy (EM) has become a common tool to pinpoint proteins involved in recombination during meiotic prophase. Each method has its advantages and disadvantages. For example, LM immunofluorescence is comparatively easier and higher throughput compared to immunogold EM localization. In addition, immunofluorescence has the advantages that a faint signal can often be enhanced by longer exposure times and colocalization using two (or more) probes with different absorbance and emission spectra is straightforward. However, immunofluorescence is not useful if the object of interest does not label with an antibody probe and is below the resolution of the LM. In comparison, immunogold EM localization is higher resolution than immunofluorescent LM localization, and individual nuclear structures, such as recombination nodules, can be identified by EM regardless of whether they are labeled or not. However, immunogold localization has other disadvantages including comparatively low signal-to-noise ratios, more difficult colocalization using gold particles of different sizes, and the inability to evaluate labeling efficiency before examining the sample using EM (a more expensive and time-consuming technique than LM). Here we describe a method that takes advantage of the good points of both immunofluorescent LM and EM to analyze two classes of late recombination nodules (RNs), only one of which labels with antibodies to MLH1 protein, a marker of crossovers. The method can be used readily with other antibodies to analyze early recombination nodules or other prophase I structures.

Meiotic Crossing Over in Maize Knob Heterochromatin.

There is ample evidence that crossing over is suppressed in heterochromatin associated with centromeres and nucleolus organizers (NORs). This characteristic has been attributed to all heterochromatin, but the generalization may not be justified. To investigate the relationship of crossing over to heterochromatin that is not associated with centromeres or NORs, we used a combination of fluorescence in situ hybridization of the maize 180-bp knob repeat to show the locations of knob heterochromatin and fluorescent immunolocalization of MLH1 protein and AFD1 protein to show the locations of MLH1 foci on maize synaptonemal complexes (SCs, pachytene chromosomes). MLH1 foci correspond to the location of recombination nodules (RNs) that mark sites of crossing over. We found that MLH1 foci occur at similar frequencies per unit length of SC in interstitial knobs and in the 1 µm segments of SC in euchromatin immediately to either side of interstitial knobs. These results indicate not only that crossing over occurs within knob heterochromatin, but also that crossing over is not suppressed in the context of SC length in maize knobs. However, because there is more DNA per unit length of SC in knobs compared to euchromatin, crossing over is suppressed (but not eliminated) in knobs in the context of DNA length compared to adjacent euchromatin.

Prescription of Hormonal Contraception by Pharmacists in Oregon: Implementation of House Bill 2879.

Prescription of hormonal contraception by pharmacists, without a doctor's visit or authorization, has been proposed as a strategy to improve access to contraception and reduce unintended pregnancy. Oregon is the first state to implement legislation expanding the scope of pharmacists to directly prescribe and dispense short-acting hormonal contraception (pill and patch). Several other states are considering similar legislation. Implementation of the policy is being researched to identify both barriers and facilitators to the successful dissemination of the practice and to determine the safety, efficacy, and acceptability of pharmacist-prescribed contraception.

Combined fluorescent and electron microscopic imaging unveils the specific properties of two classes of meiotic crossovers.

Crossovers (COs) shuffle genetic information and allow balanced segregation of homologous chromosomes during the first division of meiosis. In several organisms, mutants demonstrate that two molecularly distinct pathways produce COs. One pathway produces class I COs that exhibit interference (lowered probability of nearby COs), and the other pathway produces class II COs with little or no interference. However, the relative contributions, genomic distributions, and interactions of these two pathways are essentially unknown in nonmutant organisms because marker segregation only indicates that a CO has occurred, not its class type. Here, we combine the efficiency of light microscopy for revealing cellular functions using fluorescent probes with the high resolution of electron microscopy to localize and characterize COs in the same sample of meiotic pachytene chromosomes from wild-type tomato. To our knowledge, for the first time, every CO along each chromosome can be identified by class to unveil specific characteristics of each pathway. We find that class I and II COs have different recombination profiles along chromosomes. In particular, class II COs, which represent about 18% of all COs, exhibit no interference and are disproportionately represented in pericentric heterochromatin, a feature potentially exploitable in plant breeding. Finally, our results demonstrate that the two pathways are not independent because there is interference between class I and II COs.

Fluorescence in situ hybridization and optical mapping to correct scaffold arrangement in the tomato genome.

The order and orientation (arrangement) of all 91 sequenced scaffolds in the 12 pseudomolecules of the recently published tomato (Solanum lycopersicum, 2n = 2x = 24) genome sequence were positioned based on marker order in a high-density linkage map. Here, we report the arrangement of these scaffolds determined by two independent physical methods, bacterial artificial chromosome-fluorescence in situ hybridization (BAC-FISH) and optical mapping. By localizing BACs at the ends of scaffolds to spreads of tomato synaptonemal complexes (pachytene chromosomes), we showed that 45 scaffolds, representing one-third of the tomato genome, were arranged differently than predicted by the linkage map. These scaffolds occur mostly in pericentric heterochromatin where 77% of the tomato genome is located and where linkage mapping is less accurate due to reduced crossing over. Although useful for only part of the genome, optical mapping results were in complete agreement with scaffold arrangement by FISH but often disagreed with scaffold arrangement based on the linkage map. The scaffold arrangement based on FISH and optical mapping changes the positions of hundreds of markers in the linkage map, especially in heterochromatin. These results suggest that similar errors exist in pseudomolecules from other large genomes that have been assembled using only linkage maps to predict scaffold arrangement, and these errors can be corrected using FISH and/or optical mapping. Of note, BAC-FISH also permits estimates of the sizes of gaps between scaffolds, and unanchored BACs are often visualized by FISH in gaps between scaffolds and thus represent starting points for filling these gaps.

Preparing SC spreads with RNs for EM analysis.

Recombination nodules (RNs) are associated with synaptonemal complexes (SCs) during early prophase I of meiosis. RNs are too small to be resolved by light microscopy and can be observed directly only by electron microscopy. The patterns of RNs on SCs can be analyzed using three-dimensional reconstructions of nuclei using serial thin sections, but this method is time consuming and technically difficult. In contrast, spreads of SCs are in one plane so all RNs in each set can be visualized simultaneously, and the patterns of both early and late nodules (ENs and LNs) can be analyzed far more easily than using sections. Here, we describe methods for preparing spreads of SCs and RNs from tomato primary microsporocytes on plastic-coated slides for visualization by transmission electron microscopy (TEM).

Altered distribution of MLH1 foci is associated with changes in cohesins and chromosome axis compaction in an asynaptic mutant of tomato.

In most multicellular eukaryotes, synapsis [synaptonemal complex (SC) formation] between pairs of homologous chromosomes during prophase I of meiosis is closely linked with crossing over. Asynaptic mutants in plants have reduced synapsis and increased univalent frequency, often resulting in genetically unbalanced gametes and reduced fertility. Surprisingly, some asynaptic mutants (like as1 in tomato) have wild-type or increased levels of crossing over. To investigate, we examined SC spreads from as1/as1 microsporocytes using both light and electron microscopic immunolocalization. We observed increased numbers of MLH1 foci (a crossover marker) per unit length of SC in as1 mutants compared to wild-type. These changes are associated with reduced levels of detectable cohesin proteins in the axial and lateral elements (AE/LEs) of SCs, and the AE/LEs of as1 mutants are also significantly longer than those of wild-type or another asynaptic mutant. These results indicate that chromosome axis structure, synapsis, and crossover control are all closely linked in plants.