Induction of somatic cell haploidy by premature cell division.

Canonical mitotic and meiotic cell divisions commence with replicated chromosomes consisting of two sister chromatids. Here, we developed and explored a model of premature cell division, where nonreplicated, G0/G1-stage somatic cell nuclei are transplanted to the metaphase cytoplasm of mouse oocytes. Subsequent cell division generates daughter cells with reduced ploidy. Unexpectedly, genome sequencing analysis revealed proper segregation of homologous chromosomes, resulting in complete haploid genomes. We observed a high occurrence of somatic genome haploidization in nuclei from inbred genetic backgrounds but not in hybrids, emphasizing the importance of sequence homology between homologs. These findings suggest that premature cell division relies on mechanisms similar to meiosis I, where genome haploidization is facilitated by homologous chromosome interactions, recognition, and pairing. Unlike meiosis, no evidence of recombination between somatic cell homologs was detected. Our study offers an alternative in vitro gametogenesis approach by directly reprogramming diploid somatic cells into haploid oocytes.

Unhealthy air quality secondary to wildfires is associated with lower blastocyst yield.

OBJECTIVE: To study the impact of unhealthy air quality from the 2020 Oregon wildfires on outcomes for patients undergoing in vitro fertilization (IVF) treatment. DESIGN: A retrospective cohort study. SETTING: A university-based fertility clinic. PATIENTS: Subjects were undergoing IVF treatment from the 6 weeks preceding the wildfires through a 10-day exposure period. Cohorts were classified on the basis of whether subjects experienced patient and/or laboratory exposure to unhealthy air quality. Patient exposure was defined as at least 4 days of ovarian stimulation overlapping with the exposure, and laboratory exposure was defined as at least 2 days of IVF treatment and embryogenesis overlapping with the exposure. The unexposed cohort consisted of remaining subjects without defined exposure, with cycles in the 6 weeks preceding the wildfires. As some subjects had dual exposure and appeared in both patient and laboratory exposure cohorts, each cohort was separately compared with the unexposed control cohort. INTERVENTION: A 10-day period of unhealthy air quality caused by smoke plumes from a wildfire event. MAIN OUTCOME MEASURES: The primary outcome was the blastulation rate. Secondary outcomes included fertilization rate, number of blastocysts obtained, and cycles with no blastocysts frozen or transferred. RESULTS: Sixty-nine subjects underwent ovarian stimulation and IVF treatment during the 6 weeks preceding the wildfires through the 10-day period of unhealthy air quality. Of these, 15 patients were in the laboratory exposure cohort, 16 were in the patient exposure cohort, and 44 were unexposed. Six subjects appeared in both laboratory and patient exposure cohorts. Although neither exposure cohort had significantly decreased blastulation rate compared with the unexposed, the median number of blastocysts obtained was significantly lower in the laboratory exposure cohort than the unexposed group (2 [range 0-14] vs. 4.5 [range 0-21], respectively). The laboratory exposure cohort had significantly more cycles with no blastocysts obtained (3/15 [20%] vs. 1/44 [2%]). There were no significant differences in IVF treatment outcomes between patient exposure and unexposed cohorts. These findings persisted after controlling for age. There were no significant differences in pregnancy outcomes observed after embryo transfer between the exposure group and the unexposed group. CONCLUSION: For a cohort of patients undergoing IVF treatment, an acute episode of outside wildfire smoke exposure during fertilization and embryogenesis was associated with decreased blastocyst yield.

Strengthening the Reproductive Endocrinology and Infertility Curriculum Through Three Interactive Cases.

Introduction: Improved reproductive endocrinology and infertility (REI) curricula are needed to address educational deficiencies both at our institution and on a national level. To improve REI education for OB/GYN residents and medical students, we developed and piloted a curriculum with in-person and virtual flexibility. Methods: We developed three clinical vignettes for a facilitator-led case-based discussion among OB/GYN residents: two office cases and one emergency scenario. Cases were evaluated by content experts and tested before implementation. Pre- and postsurveys included both multiple-choice questions on content and a Likert-scale self-assessment of comfort, satisfaction, and knowledge. Postsurveys were administered immediately postintervention and at a delayed interval. Responses were compared using paired t tests and McNemar tests. Results: Eighteen learners (16 OB/GYN residents and two medical students) participated, the majority in person, of whom 17 (94%) completed a postsurvey. Self-rated proficiency in evaluating and managing irregular menses, infertility, and amenorrhea all improved significantly immediately following the intervention (p < .05 for all). Learners reported significantly more knowledge and comfort with REI compared to other subspecialties following the intervention (p < .05). More learners responded correctly to knowledge questions postintervention (p < .05 for questions 1 and 2, p = .16 for question 3). All learners were satisfied with and enjoyed the curriculum. Eight learners completed the delayed postsurvey and showed sustained improvements in knowledge and competence with REI content. Discussion: Facilitator-guided case-based learning was effective in improving learners' confidence, comfort, and knowledge in managing REI conditions, and improvements were sustained following a delayed interval.

Desire for genetically related children among transgender and gender-diverse patients seeking gender-affirming hormones.

Objective: To assess predictors of desire for genetically related children among a national cohort of reproductive-age transgender and gender-diverse patients aged 18 to 44 years initiating gender-affirming hormone therapy for the first time. Design: Cross-sectional study. Setting: National telehealth clinic. Patients: A cohort of patients from 33 US states initiating gender-affirming hormone therapy. A total of 10,270 unique transgender and gender-diverse patients-aged 18 to 44 years (median age 24 years), with no prior use of gender-affirming hormone therapy-completed clinical intake forms between September 1, 2020, and January 1, 2022. Interventions: Patient sex assigned at birth, insurance status, age, and geographic location. Main Outcome Measures: Self-reported desire for children using own genetic material. Results: Transgender and gender-diverse patients seeking gender-affirming medical treatments who are open to having genetically related children are an important population to identify and appropriately counsel. Over one quarter of the study population reported being interested in or unsure about having genetically related children, with 17.8% reporting yes and 8.4% unsure. Male-sex-assigned-at-birth patients had 1.37 (95% confidence interval: 1.25, 1.41) times higher odds of being open to having genetically related children compared with female-sex-assigned-at-birth patients. Those with private insurance had 1.13 (95% confidence interval: 1.02, 1.37) times higher odds of being open to having genetically related children compared with those without insurance. Conclusions: These findings represent the largest source of self-reported data on the desire for genetically related children among reproductive-age adult transgender and gender-diverse patients seeking gender-affirming hormones. Guidelines recommend that providers offer fertility-related counseling. These results indicate that transgender and gender-diverse patients, particularly male-sex-assigned-at-birth individuals and patients with private insurance, could benefit from counseling regarding the impacts of gender-affirming hormone therapy and gender-affirming surgeries on fertility.

Reproductive endocrinologist and infertility specialists' knowledge, skills, behaviors, and attitudes regarding the care for transgender and gender-diverse individuals.

Objective: To investigate associations between reproductive endocrinology and infertility (REI) providers' prior training and current knowledge, skills, attitudes, and behaviors regarding fertility preservation and family building for transgender and gender-diverse (T/GD) patients. Design: The survey was distributed to members of the Society for Reproductive Endocrinology and Infertility, the REI-physician-focused professional body within the American Society for Reproductive Medicine, with additional participants recruited through snowball sampling. Results: Participants (n = 206) reported on training in T/GD care; 51% endorsed prior training. Most participants (93%) believed T/GD individuals were as fit for parenthood as cisgender individuals. Prior training was associated with an increased likelihood of offering T/GD health resources and more frequent consultations with specialist colleagues.Common barriers to providing care indicated by respondents included cost, delays in gender-affirming care, and lack of knowledge of the potential impact of hormonal interventions. Common facilitators included education and training, prior experience, and affordability of services. Conclusions: Most REI providers believed T/GD individuals are fit for parenthood and agreed that prior training facilitates care for T/GD patients. The lack of provider knowledge emerged as a barrier to care. Although training helped facilitate some components of care, systemic barriers such as the cost and variability of patient population characteristics/experiences are important considerations when serving T/GD individuals.

Limitations of gene editing assessments in human preimplantation embryos.

Range of DNA repair in response to double-strand breaks induced in human preimplantation embryos remains uncertain due to the complexity of analyzing single- or few-cell samples. Sequencing of such minute DNA input requires a whole genome amplification that can introduce artifacts, including coverage nonuniformity, amplification biases, and allelic dropouts at the target site. We show here that, on average, 26.6% of preexisting heterozygous loci in control single blastomere samples appear as homozygous after whole genome amplification indicative of allelic dropouts. To overcome these limitations, we validate on-target modifications seen in gene edited human embryos in embryonic stem cells. We show that, in addition to frequent indel mutations, biallelic double-strand breaks can also produce large deletions at the target site. Moreover, some embryonic stem cells show copy-neutral loss of heterozygosity at the cleavage site which is likely caused by interallelic gene conversion. However, the frequency of loss of heterozygosity in embryonic stem cells is lower than in blastomeres, suggesting that allelic dropouts is a common whole genome amplification outcome limiting genotyping accuracy in human preimplantation embryos.

Mifepristone-misoprostol combination treatment for early pregnancy loss after embryo transfer: a case series.

Objective: Evidence strongly supports the use of mifepristone-misoprostol combination treatment for early pregnancy loss (EPL) among pregnancies conceived without assisted reproductive technologies. No literature exists, however, regarding the efficacy of this treatment in the medical management of EPL among pregnancies after in vitro fertilization and embryo transfer (IVF-ET). These patients differ as some use exogenous hormonal supplementation to provide pregnancy support. Thus, the management for EPL may differ between unassisted conceptions and those after ET. Mifepristone, a progesterone receptor antagonist, may demonstrate an altered treatment effect when used with misoprostol to manage EPL in assisted reproductive technologie-conceived pregnancies. Objective: To describe our institution's experience using mifepristone-misoprostol to manage EPL after in vitro fertilization with embryo transfer IVF-ET. Design: Retrospective case series. Setting: Single academic institution from 2020 to 2022. Patientss: Nine patients with ultrasound confirmed EPL after IVF-ET. Interventions: All 9 patients underwent in vitro fertilization followed by fresh or frozen embryo transfer. All 9 received 200 mg of mifepristone 24 hours before 800 µg of misoprostol. Main Outcome Measurements: Incomplete abortion, need for surgical management, number of days to negative serum human chorionic gonadotropin (hCG). Results: Of the 9 subjects included, one had a programmed frozen embryo transfer cycle, 6 had modified natural frozen embryo transfer cycles, and 2 underwent fresh ET. Eight subjects had successful expulsion of tissue with one dose of treatment, and one required uterine aspiration. No subjects required additional dosing of misoprostol. The mean number of days elapsed from mifepristone treatment to tissue expulsion was 4.89 ± 11.30 days and the mean days to negative-range serum hCG was 36.89 ± 18.59 days. At the initial ultrasound, all pregnancies had one gestational sac seen; 5/9 had a yolk sac; only 3 had fetal cardiac activity. The mean gestational age at the time of EPL diagnosis was 55.22 ± 8.77 days, with the majority (8/9) having completed 7 weeks gestation. Conclusions: Mifepristone-misoprostol combination treatment appears to be a reasonable option for those with EPL after IVF-ET. Future, larger-scale studies are needed comparing combination treatment with misoprostol only among various ET protocols.

Patients with Recurrent Pregnancy Loss Have Similar Embryonic Preimplantation Genetic Testing Aneuploidy Rates and In Vitro Fertilization Outcomes to Infertility Patients.

Objective: To evaluate aneuploidy rates and in vitro fertilization (IVF)/pregnancy outcomes for patients undergoing IVF and preimplantation genetic testing for aneuploidy (PGT-A) with a recurrent pregnancy loss (RPL) diagnosis compared to infertility diagnoses without RPL. Design: Retrospective cohort study. Setting: Academic fertility center. Patients: Of 372 patients undergoing IVF/PGT-A between January 2016-December 2018, 294 patients were included in the analysis: 56 patients with an RPL diagnosis and 238 with infertility diagnoses without RPL. Interventions: None. Main Outcome Measures: The primary outcome measured was the embryonic aneuploidy rate. Secondary outcomes included fertilization and blastulation rates, number of blastocysts biopsied, cycles without euploid blastocysts, and rates of pregnancy losses, clinical pregnancies, and live births after a euploid embryo transfer. Results: The cohort included 56 patients with RPL and 238 patients without RPL, including data from their first IVF cycle within the time period. Aneuploidy rates were similar between the groups, with a mean of 55% (±31%) in RPL and 54% (±34%) in non-RPL cycles. Similar rates persisted after controlling for age, ovarian reserve, and infertility diagnosis. Fertilization and blastulation rates, as well as cumulative clinical pregnancy, pregnancy loss, and live birth rates after the transfer of at least one euploid embryo were also similar between the two groups. Conclusions: These results suggest that IVF/PGT-A cycles from patients with an RPL diagnosis have similar IVF and pregnancy outcomes to those of patients with infertility without RPL. This research can help guide counseling for RPL patients considering IVF with PGT-A.

Horizontal mtDNA transfer between cells is common during mouse development.

Cells transmit their genomes vertically to daughter cells during cell divisions. Here, we demonstrate the occurrence and extent of horizontal mitochondrial (mt)DNA acquisition between cells that are not in a parent-offspring relationship. Extensive single-cell sequencing from various tissues and organs of adult chimeric mice composed of cells carrying distinct mtDNA haplotypes showed that a substantial fraction of individual cardiomyocytes, neurons, glia, intestinal, and spleen cells captured donor mtDNA at high levels. In addition, chimeras composed of cells with wild-type and mutant mtDNA exhibited increased trafficking of wild-type mtDNA to mutant cells, suggesting that horizontal mtDNA transfer may be a compensatory mechanism to restore compromised mitochondrial function. These findings establish the groundwork for further investigations to identify mtDNA donor cells and mechanisms of transfer that could be critical to the development of novel gene therapies.

Changing gender gap and practice patterns in reproductive endocrinology and infertility subspecialists in the United States: a Society for Reproductive Endocrinology and Infertility report.

OBJECTIVE: To identify changes in current practice patterns, salaries, and satisfaction by gender and by years in practice among board-certified reproductive endocrinology and infertility (REI) subspecialists in the United States. DESIGN: Cross-sectional web-based survey including 37 questions conducted by the Society for Reproductive Endocrinology and Infertility. SETTING: Not applicable. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The primary outcome measures were total compensation and practice patterns compared by gender and the type of practice. The secondary outcomes included demographics, the number of in vitro fertilization cycles, surgeries performed, and the morale of survey respondents. RESULT(S): There were 370 respondents (48.4% women and 51.4% men). Compared with a similar survey conducted 6 years earlier, a 27% increase in the number of female respondents was observed in this survey. There was a marginally significant trend toward lower compensation for female than male REI subspecialists (17% lower, $472,807 vs. $571,969). The gap was seen for responders with ≥10 years' experience, which is also when there was the largest gap between private and academic practice (mean $820,997 vs, $391,600). Most (77%) felt positively about the current state of the reproductive endocrinology field, and >90% would choose the subspecialty again. CONCLUSION(S): There has been a substantial increase in the number of recent female REI subspecialists showing less disparity in compensation, and the gap appears to be closing. There is an increasing gap in compensation between private and academic practices with ≥5 years of experience. Reproductive endocrinology and infertility remains a high morale specialty.

Haploidy in somatic cells is induced by mature oocytes in mice.

Haploidy is naturally observed in gametes; however, attempts of experimentally inducing haploidy in somatic cells have not been successful. Here, we demonstrate that the replacement of meiotic spindles in mature metaphases II (MII) arrested oocytes with nuclei of somatic cells in the G0/G1 stage of cell cycle results in the formation of de novo spindles consisting of somatic homologous chromosomes comprising of single chromatids. Fertilization of such oocytes with sperm triggers the extrusion of one set of homologous chromosomes into the pseudo-polar body (PPB), resulting in a zygote with haploid somatic and sperm pronuclei (PN). Upon culture, 18% of somatic-sperm zygotes reach the blastocyst stage, and 16% of them possess heterozygous diploid genomes consisting of somatic haploid and sperm homologs across all chromosomes. We also generate embryonic stem cells and live offspring from somatic-sperm embryos. Our finding may offer an alternative strategy for generating oocytes carrying somatic genomes.

Home collection of products of conception: Can chromosomal analysis be obtained?

INTRODUCTION: The majority of early pregnancy losses are due to chromosomal abnormalities, which can be evaluated by testing the products of conception (POCs). Traditionally, a dilation and curettage (D&C) is recommended to patients to obtain suitable tissue for analysis. This case series sought to determine whether patients with first-trimester pregnancy losses can successfully obtain analysis of POCs if they pass tissue with expectant management or misoprostol, rather than D&C. MATERIALS AND METHODS: This is a retrospective cohort study of patients who desired analysis of POCs following an early pregnancy loss. The POCs were collected either at home by patients, or by providers following a D&C, and sent to an outside laboratory for single nucleotide polymorphism (SNP) microarray analysis. RESULTS: Of 50 identified patients, chromosomal analysis was successfully completed on: 22/34 (65%) of patients who passed pregnancy with misoprostol, 14/14 (100%) with D&C, and 1/2 (50%) with expectant management (x2, p = 0.03). Analysis was successful up to 9 days after tissue was collected, and 55 days after diagnosis of loss. CONCLUSION: In our study, D&C was the superior method to obtain POCs for chromosomal analysis following a miscarriage, but medical and expectant management still had clinically acceptable success rates as well. Although further study is indicated, this small case series suggests that patient counselling regarding miscarriage management and desired chromosomal analysis should continue to include misoprostol and expectant management as inferior but still appropriate alternatives to D&C.

Use of gestational surrogates for women with Eisenmenger syndrome: a cost-effectiveness analysis.

Objective: Eisenmenger syndrome (ES) is regarded as a contraindication to pregnancy, with therapeutic abortion recommended in the event of unintended pregnancy. However, women with ES continue to desire and attempt pregnancy despite grave risks to their own health. This study compares the costs and outcomes of pregnancy in women with ES to the use of gestational surrogates in their pregnancies.Study design: A decision-analytic model was built using TreeAge software that compared use of gestational surrogates and pregnancy in women with ES. Maternal death and neonatal outcomes including intrauterine fetal demise, preterm birth, cerebral palsy, and death were assessed. All probabilities and costs were derived from the literature. Utilities were discounted at a rate of 3% across the expected lifespan to generate quality-adjusted life years (QALYs). Univariate and multivariate sensitivity analyses were performed to evaluate the robustness of the model given changes in baseline assumptions.Results: In a theoretical cohort of 1000 women with ES, pregnancy would result in 360 maternal deaths, 100 stillbirths, 477 preterm births, and 157 neonatal deaths . In these highly desired pregnancies, use of gestational surrogates would prevent 99 and 98% of maternal and neonatal death, respectively. Cases and costs of preterm birth and associated cerebral palsy are also significantly reduced. Use of a gestational surrogate would save $518,255 per woman with a gain of 6.77 QALYs, a dominant strategy. The approach is cost-effective up to a cost of surrogacy of $1.2 million and even if the surrogate achieves pregnancy only 30% of the time.Conclusions: The use of surrogate mothers for those with ES is cost-effective and results in significantly improved maternal and neonatal outcomes. These benefits are robust in the face of high surrogacy costs largely due to the marked reduction in maternal mortality and preterm birth. These findings should be used to underscore the importance of broadening health care financing for medically-indicated assisted reproduction.

Germline transmission of donor, maternal and paternal mtDNA in primates.

STUDY QUESTION: What are the long-term developmental, reproductive and genetic consequences of mitochondrial replacement therapy (MRT) in primates? SUMMARY ANSWER: Longitudinal investigation of MRT rhesus macaques (Macaca mulatta) generated with donor mtDNA that is exceedingly distant from the original maternal counterpart suggest that their growth, general health and fertility is unremarkable and similar to controls. WHAT IS KNOWN ALREADY: Mitochondrial gene mutations contribute to a diverse range of incurable human disorders. MRT via spindle transfer in oocytes was developed and proposed to prevent transmission of pathogenic mtDNA mutations from mothers to children. STUDY DESIGN, SIZE, DURATION: The study provides longitudinal studies on general health, fertility as well as transmission and segregation of parental mtDNA haplotypes to various tissues and organs in five adult MRT rhesus macaques and their offspring. PARTICIPANTS/MATERIALS, SETTING, METHODS: MRT was achieved by spindle transfer between metaphase II oocytes from genetically divergent rhesus macaque populations. After fertilization of oocytes with sperm, heteroplasmic zygotes contained an unequal mixture of three parental genomes, i.e. donor (≥97%), maternal (≤3%), and paternal (≤0.1%) mitochondrial (mt)DNA. MRT monkeys were grown to adulthood and their development and general health was regularly monitored. Reproductive fitness of male and female MRT macaques was evaluated by time-mated breeding and production of live offspring. The relative contribution of donor, maternal, and paternal mtDNA was measured by whole mitochondrial genome sequencing in all organs and tissues of MRT animals and their offspring. MAIN RESULTS AND THE ROLE OF CHANCE: Both male and female MRT rhesus macaques containing unequal mixture of three parental genomes, i.e. donor (≥97%), maternal (≤3%), and paternal (≤0.1%) mtDNA reached healthy adulthood, were fertile and most animals stably maintained the initial ratio of parental mtDNA heteroplasmy and donor mtDNA was transmitted from females to offspring. However, in one monkey out of four analyzed, initially negligible maternal mtDNA heteroplasmy levels increased substantially up to 17% in selected internal tissues and organs. In addition, two monkeys showed paternal mtDNA contribution up to 33% in selected internal tissues and organs. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Conclusions in this study were made on a relatively low number of MRT monkeys, and on only one F1 (first generation) female. In addition, monkey MRT involved two wildtype mtDNA haplotypes, but not disease-relevant variants. Clinical trials on children born after MRT will be required to fully determine safety and efficacy of MRT for humans. WIDER IMPLICATIONS OF THE FINDINGS: Our data show that MRT is compatible with normal postnatal development including overall health and reproductive fitness in nonhuman primates without any detected adverse effects. 'Mismatched' donor mtDNA in MRT animals even from the genetically distant mtDNA haplotypes did not cause secondary mitochondrial dysfunction. However, carry-over maternal or paternal mtDNA contributions increased substantially in selected internal tissues / organs of some MRT animals implying the possibility of mtDNA mutation recurrence. STUDY FUNDING/COMPETING INTEREST(S): This work has been funded by the grants from the Burroughs Wellcome Fund, the National Institutes of Health (RO1AG062459 and P51 OD011092), National Research Foundation of Korea (2018R1D1A1B07043216) and Oregon Health & Science University institutional funds. The authors declare no competing interests.

Deleterious mtDNA mutations are common in mature oocytes.

Heritable mitochondrial DNA (mtDNA) mutations are common, yet only a few recurring pathogenic mtDNA variants account for the majority of known familial cases in humans. Purifying selection in the female germline is thought to be responsible for the elimination of most harmful mtDNA mutations during oogenesis. Here we show that deleterious mtDNA mutations are abundant in ovulated mature mouse oocytes and preimplantation embryos recovered from PolG mutator females but not in their live offspring. This implies that purifying selection acts not in the maternal germline per se, but during post-implantation development. We further show that oocyte mtDNA mutations can be captured and stably maintained in embryonic stem cells and then reintroduced into chimeras, thereby allowing examination of the effects of specific mutations on fetal and postnatal development.

Author Correction: Mitochondrial replacement in human oocytes carrying pathogenic mitochondrial DNA mutations.

Change history In this Letter, there are several errors regarding the assignments of mtDNA haplotypes for a subset of egg donors from our study. These errors have not been corrected online.