Error-prone repair of stalled replication forks drives mutagenesis and loss of heterozygosity in haploinsufficient BRCA1 cells.

Germline mutations in the BRCA genes are associated with a higher risk of carcinogenesis, which is linked to an increased mutation rate and loss of the second unaffected BRCA allele (loss of heterozygosity, LOH). However, the mechanisms triggering mutagenesis are not clearly understood. The BRCA genes contain high numbers of repetitive DNA sequences. We detected replication forks stalling, DNA breaks, and deletions at these sites in haploinsufficient BRCA cells, thus identifying the BRCA genes as fragile sites. Next, we found that stalled forks are repaired by error-prone pathways, such as microhomology-mediated break-induced replication (MMBIR) in haploinsufficient BRCA1 breast epithelial cells. We detected MMBIR mutations in BRCA1 tumor cells and noticed deletions-insertions (>50 bp) at the BRCA1 genes in BRCA1 patients. Altogether, these results suggest that under stress, error-prone repair of stalled forks is upregulated and induces mutations, including complex genomic rearrangements at the BRCA genes (LOH), in haploinsufficient BRCA1 cells.

Efficient direct reprogramming of mature amniotic cells into endothelial cells by ETS factors and TGFß suppression.

ETS transcription factors ETV2, FLI1, and ERG1 specify pluripotent stem cells into induced vascular endothelial cells (iVECs). However, iVECs are unstable and drift toward nonvascular cells. We show that human midgestation c-Kit(-) lineage-committed amniotic cells (ACs) can be reprogrammed into vascular endothelial cells (rAC-VECs) without transitioning through a pluripotent state. Transient ETV2 expression in ACs generates immature rAC-VECs, whereas coexpression with FLI1/ERG1 endows rAC-VECs with a vascular repertoire and morphology matching mature endothelial cells (ECs). Brief TGFß-inhibition functionalizes VEGFR2 signaling, augmenting specification of ACs into rAC-VECs. Genome-wide transcriptional analyses showed that rAC-VECs are similar to adult ECs in which vascular-specific genes are expressed and nonvascular genes are silenced. Functionally, rAC-VECs form stable vasculature in Matrigel plugs and regenerating livers. Therefore, short-term ETV2 expression and TGFß inhibition with constitutive ERG1/FLI1 coexpression reprogram mature ACs into durable rAC-VECs with clinical-scale expansion potential. Banking of HLA-typed rAC-VECs establishes a vascular inventory for treatment of diverse disorders.

Endothelial-derived angiocrine signals induce and sustain regenerative lung alveolarization.

To identify pathways involved in adult lung regeneration, we employ a unilateral pneumonectomy (PNX) model that promotes regenerative alveolarization in the remaining intact lung. We show that PNX stimulates pulmonary capillary endothelial cells (PCECs) to produce angiocrine growth factors that induce proliferation of epithelial progenitor cells supporting alveologenesis. Endothelial cells trigger expansion of cocultured epithelial cells, forming three-dimensional angiospheres reminiscent of alveolar-capillary sacs. After PNX, endothelial-specific inducible genetic ablation of Vegfr2 and Fgfr1 in mice inhibits production of MMP14, impairing alveolarization. MMP14 promotes expansion of epithelial progenitor cells by unmasking cryptic EGF-like ectodomains that activate the EGF receptor (EGFR). Consistent with this, neutralization of MMP14 impairs EGFR-mediated alveolar regeneration, whereas administration of EGF or intravascular transplantation of MMP14(+) PCECs into pneumonectomized Vegfr2/Fgfr1-deficient mice restores alveologenesis and lung inspiratory volume and compliance function. VEGFR2 and FGFR1 activation in PCECs therefore increases MMP14-dependent bioavailability of EGFR ligands to initiate and sustain alveologenesis.

Timing of Testicular Biopsy in Relation to Oocyte Retrieval and the Outcomes of Intracytoplasmic Sperm Injection.

PURPOSE: We evaluate microscopic (micro) testicular sperm extraction (TESE) timing relative to oocyte retrieval on intracytoplasmic sperm injection outcome. MATERIALS AND METHODS: Couples with nonobstructive azoospermia who underwent intracytoplasmic sperm injection with freshly retrieved spermatozoa were analyzed based on whether micro-TESE was performed at least 1 day prior to oocyte retrieval (TESE-day-before group) or on the day of oocyte retrieval (TESE-day-of group). Embryology and clinical outcomes were compared. RESULTS: The percentage of patients who underwent a successful testicular sperm retrieval was significantly lower in the TESE-day-before cohort (62%) than in the TESE-day-of cohort (69%; odds ratio [OR] 1.4, 95% CI [1.1, 1.7], P < .001). The fertilization rate was also found to be significantly lower in the TESE-day-before group (45%) than in the TESE-day-of group (53%; OR 1.4, 95% CI [1.2, 1.7], P = .01). Although the association between the cleavage rate and TESE timing was not statistically significant, the implantation rate was found to be significantly higher in the day-before cohort (28%) than in the day-of cohort (22%; OR 0.7, 95% CI [0.6, 0.9], P = .01). Nevertheless, it was found that the clinical pregnancy and delivery rates were not statistically significantly associated with the TESE timing. CONCLUSIONS: Although sperm retrieval and fertilization rates were lower in the TESE-day-before cohort, the 2 cohorts showed comparable embryologic and clinical outcomes. Micro-TESE can be performed before oocyte harvesting to provide physicians ample time to decide between cancelling oocyte retrieval or retrieving oocytes for cryopreservation.

Pluripotent stem cell-derived epithelium misidentified as brain microvascular endothelium requires ETS factors to acquire vascular fate.

Cells derived from pluripotent sources in vitro must resemble those found in vivo as closely as possible at both transcriptional and functional levels in order to be a useful tool for studying diseases and developing therapeutics. Recently, differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cells (ECs) with blood-brain barrier (BBB)-like properties has been reported. These cells have since been used as a robust in vitro BBB model for drug delivery and mechanistic understanding of neurological diseases. However, the precise cellular identity of these induced brain microvascular endothelial cells (iBMECs) has not been well described. Employing a comprehensive transcriptomic metaanalysis of previously published hPSC-derived cells validated by physiological assays, we demonstrate that iBMECs lack functional attributes of ECs since they are deficient in vascular lineage genes while expressing clusters of genes related to the neuroectodermal epithelial lineage (Epi-iBMEC). Overexpression of key endothelial ETS transcription factors (ETV2, ERG, and FLI1) reprograms Epi-iBMECs into authentic endothelial cells that are congruent with bona fide endothelium at both transcriptomic as well as some functional levels. This approach could eventually be used to develop a robust human BBB model in vitro that resembles the human brain EC in vivo for functional studies and drug discovery.

Genetic profiling of azoospermic men to identify the etiology and predict reproductive potential.

PURPOSE: To identify germline mutations related to azoospermia etiology and reproductive potential of surgically retrieved spermatozoa, and to investigate the feasibility of predicting seminiferous tubule function of nonobstructive azoospermic men by transcriptomic profiling of ejaculates. MATERIALS AND METHODS: Sperm specimens were obtained from 30 men (38.4 ± 6 years) undergoing epididymal sperm aspiration for obstructive azoospermia (OA, n = 19) acquired by vasectomy, or testicular biopsy for nonobstructive azoospermia (NOA, n = 11). To evaluate for a correlation with azoospermia etiology, DNAseq was performed on surgically retrieved spermatozoa, and cell-free RNAseq on seminal fluid (n = 23) was performed to predict spermatogenesis in the seminiferous tubule. RESULTS: Overall, surgically retrieved sperm aneuploidy rates were 1.7% and 1.8% among OA and NOA cohorts, respectively. OA men carried housekeeping-related gene mutations, while NOA men displayed mutations on genes involved in crucial spermiogenic functions (AP1S2, AP1G2, APOE). We categorized couples within each cohort according to ICSI clinical outcomes to investigate genetic causes that may affect reproductive potential. All OA-fertile men (n = 9) carried mutations in ZNF749 (sperm production), whereas OA-infertile men (n = 10) harbored mutations in PRB1, which is essential for DNA replication. NOA-fertile men (n = 8) carried mutations in MPIG6B (stem cell lineage differentiation), whereas NOA-infertile individuals (n = 3) harbored mutations in genes involved in spermato/spermio-genesis (ADAM29, SPATA31E1, MAK, POLG, IFT43, ATG9B) and early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC8, REPIN1, MAPRE3, ARL4C). Transcriptomic assessment of cell-free RNAs in seminal fluid from NOA men allowed the prediction of residual spermatogenic foci. CONCLUSIONS: Sperm genome profiling provides invaluable information on azoospermia etiology and identifies gene-related mechanistic links to reproductive performance. Moreover, RNAseq assessment of seminal fluid from NOA men can help predict sperm retrieval during testicular biopsies.

Xenograft model of heterotopic transplantation of human ovarian cortical tissue and its clinical relevance.

In brief: Xenografts of human ovarian cortical tissue provide a tractable model of heterotopic autotransplantation that is used for fertility preservation in patients undergoing ablative chemo/radiotherapy. This study describes the behavior of hundreds of xenografts to establish a framework for the clinical function of ovarian cortex following autotransplantation over short- and long-term intervals. Abstract: More than 200 live births have been achieved using autotransplantation of cryopreserved ovarian cortical fragments, yet challenges remain to be addressed. Ischemia of grafted tissue undermines viability and longevity, typically requiring transplantation of multiple cortical pieces; and the dynamics of recruitment within a graft and the influence of parameters like size and patient age at the time of cryopreservation are not well-defined. Here, we describe results from a series of experiments in which we xenografted frozen/thawed human ovarian tissue (n = 440) from 28 girls and women (age range 32 weeks gestational age to 46 years, median 24.3 ± 4.6). Xenografts were recovered across a broad range of intervals (1-52 weeks post-transplantation) and examined histologically to quantify follicle density and distribution. The number of antral follicles in xenografted cortical fragments correlated positively with the total follicle number and was significantly reduced with increased patient age. Within xenografts, follicles were distributed in focal clusters, similar to the native ovary, but the presence of a leading antral follicle coincided with increased proliferation of surrounding follicles. These results underscore the importance of transplanting ovarian tissue with a high density of follicles and elucidate a potential paracrine influence of leading antral follicles on neighboring follicles of earlier stages. This temporal framework for interpreting the kinetics of follicle growth/mobilization may be useful in setting expectations and guiding the parameters of clinical autotransplantation.

Conversion of adult endothelium to immunocompetent haematopoietic stem cells.

Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully reprogramming adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb, Gfi1, Runx1, and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors. The induction phase (days 0-8) of conversion is initiated by expression of FGRS in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (days 8-20), RUNX1+ FGRS-transduced endothelial cells commit to a haematopoietic fate, yielding rEC-HSCs that no longer require FGRS expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (days 20-28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, and can be used for clonal engraftment and serial primary and secondary multi-lineage reconstitution, including antigen-dependent adaptive immune function. Inhibition of TGFß and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Pluripotency-independent conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders.

A novel approach toward the generation of oocytes by direct diploid cell haploidization.

Oocyte-mediated somatic cell haploidization is a process in which a diploid cell halves its chromosomal content by segregating its homologue within the ooplasm. Replacing the donor oocyte nucleus with a patient's female diploid somatic nucleus can generate patient-genotyped oocytes. Insemination of these resulting constructs enables their activation and induces a reductive meiotic division, haploidizing the diploid female donor cell that can subsequently support syngamy with the male genome and create a zygote. So far, experimental data for this method have been limited and have not consistently proven the generation of chromosomally normal embryos. Overall, we achieved reconstruction of murine oocytes with a micromanipulation survival rate of 56.5%, and a correct haploidization and fertilization rate of 31.2%, resulting in a 12.7% blastocyst rate. Time-lapse analysis revealed that reconstructed embryos underwent a timely polar body extrusion and pronuclear appearance followed by a satisfactory embryonic cleavage, comparable with the control. Whole genome sequencing of the analyzed embryos indicated that 27.3% (6/22) were properly diploid. Our findings suggest that diploid cell haploidization may be a feasible technique for creating functional gametes in mammals.

Effects of Chemo- and Radiation Therapy on Microsurgical Testicular Sperm Extraction for Men with Nonobstructive Azoospermia.

PURPOSE: Men who survive cancer as children or young adults may have severe spermatogenic impairment with azoospermia requiring surgical sperm retrieval and assisted reproductive technologies. We assessed treatment outcomes from a large series of cancer patients with prior radiation and/or chemotherapy. MATERIALS AND METHODS: Men with nonobstructive azoospermia who underwent initial microsurgical testicular sperm extraction from 1995-2020 from a high-volume surgeon at a single institution were identified. Those with a history of malignancy treated by radiation therapy and/or chemotherapy were included. The primary outcome was successful sperm retrieval. RESULTS: A total of 106 men were evaluated, of whom 57 received chemotherapy and radiation, 44 received only chemotherapy and 5 received only radiation. Sperm retrieval was successful in 39 of 106 (37%) men, with higher likelihood of retrieval in men who received only chemotherapy compared to men who received chemotherapy and radiation (61% vs 18%, p <0.001). None of the 18 patients who received chemotherapy with radiation to the pelvis had successful sperm retrieval, compared to 26% of patients who received chemotherapy with extra-pelvic radiation (p=0.02). CONCLUSIONS: Chemotherapy and radiation for cancer may result in nonobstructive azoospermia that can be treated to allow fertility. However, pelvic radiation therapy is associated with the worst prognosis for successful treatment with microsurgical sperm retrieval and in vitro fertilization; we observed no cases of successful retrieval in men who received pelvic radiation therapy. These data are useful for pretreatment counseling, suggesting that men with prior radiation therapy may not be candidates for surgical sperm retrieval.

Age-specific blastocyst conversion rates in embryo cryopreservation cycles.

RESEARCH QUESTION: What is the blastocyst conversion rate in embryo cryopreservation cycles, per year of female age? DESIGN: Retrospective cohort study including patients undergoing their first ovarian stimulation cycle at our center with planned freeze-all strategy January 1st, 2014-June 30th, 2020. Primary outcome was blastocyst conversion rate. Secondary outcomes included mature oocyte and fertilization rates. Patients were stratified by year of age to assess oocyte yield and embryo development outcomes. RESULTS: 3,362 patients were included. The median blastocyst conversion rate in patients ≤30 was 66.7% (interquartile range 50.0-86.6) and remained statistically comparable through age 40 with a significant decline among ages ≥41 (41-years: marginal effect (ME) -5.2% (-9.7 to -0.7); 42-years: ME -9.6% (-14.3 to -4.8); 43-years: ME -7.7% (-12.8 to -2.6); ≥44-years: ME -20.8% (-26.5 to -15.1)). For the entire cohort, the median mature oocyte rate was 81.8% and the median fertilization rate was 81.8%. The mature oocyte and fertilization rates remained statistically comparable for each year of age except age ≥44 which had a statistically significantly increased mature oocyte rate (ME 4.4% (1.3 to 7.5)) and statistically significantly decreased fertilization rate (ME -5.8% (-9.8 to -1.9)) CONCLUSIONS: In embryo cryopreservation cycles, the blastocyst conversion rate remained statistically comparable through age 40 followed by a statistically significant decline for patients ≥41; however, the mature oocyte and fertilization rates were not impacted by increasing age until age ≥44. Even in women ≥44, over 40% of fertilized oocytes developed to blastocyst. Overall, this information is useful when counseling patients during the embryo culture stage regarding predicted blastocyst yield.

Comparison of fresh and frozen ejaculated spermatozoa in sibling oocyte recipient cycles.

RESEARCH QUESTION: Do IVF and intracytoplasmic sperm injection cycles using fresh and frozen ejaculated spermatozoa result in similar pregnancy outcomes in couples with non-male factor infertility? DESIGN: Retrospective cohort study; patients undergoing donor egg recipient cycles, in which oocytes from a single ovarian stimulation were split between two recipients, were reviewed. Two recipients of oocytes from a single donor were paired and categorized based on the type of ejaculated spermatozoa (fresh/frozen). Outcomes included delivery rate, implantation, pregnancy, pregnancy loss and fertilization rates. RESULTS: Of the 408 patients who received oocytes from a split donor oocyte cycle, 45 pairs of patients used discrepant types of ejaculated spermatozoa and were included in the study. Fertilization rate: fresh (74.8%); frozen (68.6%) (P = 0.13). Pregnancy rate: fresh (76%); frozen (67%); delivery rate: fresh (69%); frozen (44%); implantation rate was significantly higher: fresh (64%); frozen (36%) (P = 0.04). Rate of pregnancy loss was significantly higher in the frozen group compared with the fresh group (33% versus 5.9%, P = 0.013). Adjusted odds for delivery was 67% lower in the frozen group (95% CI 0.12, 0.89). Adjusted odds of pregnancy (adjusted OR 0.67, 95% CI 0.20, 2.27) and implantation (adjusted OR 0.5, 95% CI 0.12, 2.12) were not significantly different between the frozen and fresh sperm groups. CONCLUSION: In this model that controls for oocyte quality by using paired recipients from the same donor, frozen ejaculated spermatozoa resulted in lower delivery rates than those using fresh spermatozoa.

The DNA replication program is altered at the FMR1 locus in fragile X embryonic stem cells.

Fragile X syndrome (FXS) is caused by a CGG repeat expansion in the FMR1 gene that appears to occur during oogenesis and during early embryogenesis. One model proposes that repeat instability depends on the replication fork direction through the repeats such that (CNG)n hairpin-like structures form, causing DNA polymerase to stall and slip. Examining DNA replication fork progression on single DNA molecules at the endogenous FMR1 locus revealed that replication forks stall at CGG repeats in human cells. Furthermore, replication profiles of FXS human embryonic stem cells (hESCs) compared to nonaffected hESCs showed that fork direction through the repeats is altered at the FMR1 locus in FXS hESCs, such that predominantly the CCG strand serves as the lagging-strand template. This is due to the absence of replication initiation that would typically occur upstream of FMR1, suggesting that altered replication origin usage combined with fork stalling promotes repeat instability during early embryonic development.

Ovarian Reserve Disorders, Can We Prevent Them? A Review.

The ovarian reserve is finite and begins declining from its peak at mid-gestation until only residual follicles remain as women approach menopause. Reduced ovarian reserve, or its extreme form, premature ovarian insufficiency, stems from multiple factors, including developmental, genetic, environmental exposures, autoimmune disease, or medical/surgical treatment. In many cases, the cause remains unknown and resulting infertility is not ultimately addressed by assisted reproductive technologies. Deciphering the mechanisms that underlie disorders of ovarian reserve could improve the outcomes for patients struggling with infertility, but these disorders are diverse and can be categorized in multiple ways. In this review, we will explore the topic from a perspective that emphasizes the prevention or mitigation of ovarian damage. The most desirable mode of fertoprotection is primary prevention (intervening before ablative influence occurs), as identifying toxic influences and deciphering the mechanisms by which they exert their effect can reduce or eliminate exposure and damage. Secondary prevention in the form of screening is not recommended broadly. Nevertheless, in some instances where a known genetic background exists in discrete families, screening is advised. As part of prenatal care, screening panels include some genetic diseases that can lead to infertility or subfertility. In these patients, early diagnosis could enable fertility preservation or changes in family-building plans. Finally, Tertiary Prevention (managing disease post-diagnosis) is critical. Reduced ovarian reserve has a major influence on physiology beyond fertility, including delayed/absent puberty or premature menopause. In these instances, proper diagnosis and medical therapy can reduce adverse effects. Here, we elaborate on these modes of prevention as well as proposed mechanisms that underlie ovarian reserve disorders.

Wnt inhibition promotes vascular specification of embryonic cardiac progenitors.

Several studies have demonstrated a multiphasic role for Wnt signaling during embryonic cardiogenesis and developed protocols that enrich for cardiac derivatives during in vitro differentiation of human pluripotent stem cells (hPSCs). However, few studies have investigated the role of Wnt signaling in the specification of cardiac progenitor cells (CPCs) toward downstream fates. Using transgenic mice and hPSCs, we tracked endothelial cells (ECs) that originated from CPCs expressing NKX2.5. Analysis of EC-fated CPCs at discrete phenotypic milestones during hPSC differentiation identified reduced Wnt activity as a hallmark of EC specification, and the enforced activation or inhibition of Wnt reduced or increased, respectively, the degree of vascular commitment within the CPC population during both hPSC differentiation and mouse embryogenesis. Wnt5a, which has been shown to exert an inhibitory influence on Wnt signaling during cardiac development, was dynamically expressed during vascular commitment of hPSC-derived CPCs, and ectopic Wnt5a promoted vascular specification of hPSC-derived and mouse embryonic CPCs.

Length of estradiol exposure >100 pg/ml in the follicular phase affects pregnancy outcomes in natural frozen embryo transfer cycles.

STUDY QUESTION: Do the length of follicular phase estradiol exposure and the total length of the follicular phase affect pregnancy and live birth outcomes in natural frozen embryo transfer (FET) cycles? SUMMARY ANSWER: An estradiol level >100 pg/ml for ≤4 days including the LH surge day is associated with worse pregnancy and live birth outcomes; however, the total length of the follicular phase is not associated with pregnancy and live birth outcomes. WHAT IS KNOWN ALREADY: An estradiol level that increases above 100 pg/ml and continues to increase is indicative of the selection and development of a dominant follicle. In programmed FET cycles, a limited duration of follicular phase estradiol of <9 days results in worse pregnancy rates, but a prolonged exposure to follicular phase estradiol for up to 4 weeks does not affect pregnancy outcomes. It is unknown how follicular phase characteristics affect pregnancy outcomes in natural FET cycles. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study included infertile patients in an academic hospital setting who underwent their first natural frozen autologous Day-5 embryo transfer cycle in our IVF clinic between 01 January 2013 and 31 December 2018. Donor oocyte and gestational carrier cycles were excluded. PARTICIPANTS/MATERIALS, SETTING, METHODS: The primary outcomes of this study were pregnancy and live birth rates. Patients were stratified into two groups based on the cohorts' median number of days from the estradiol level of >100 pg/ml before the LH surge: Group 1 (≤4 days; n = 1052 patients) and Group 2 (>4 days; n = 839 patients). Additionally, patients were stratified into two groups based on the cohorts' median cycle day of LH surge: Group 1 (follicular length ≤15 days; n = 1287 patients) and Group 2 (follicular length >15 days; n = 1071 patients). A subgroup analysis of preimplantation genetic testing for aneuploidies (PGT-A) embryo transfer cycles was performed. Logistic regression analysis, adjusted a priori for patient age, number of embryos transferred, and use of PGT-A, was used to estimate the odds ratio (OR) with a 95% CI. MAIN RESULTS AND THE ROLE OF CHANCE: In the length of elevated estradiol analysis, the pregnancy rate per embryo transfer was statistically significantly lower in patients with an elevated estradiol to surge of ≤4 days (65.6%) compared to patients with an elevated estradiol to surge of >4 days (70.9%; OR 1.30 (95% CI 1.06-1.58)). The live birth rate per embryo transfer was also statistically significantly lower in patients with an elevated estradiol to surge of ≤4 days (46.6%) compared to patients with an elevated estradiol to surge of >4 days (52.0%; OR 1.23 (95% CI 1.02-1.48)). In the follicular phase length analysis, the pregnancy rate per embryo transfer was similar between patients with a follicular length of ≤15 days (65.4%) and patients with a follicular length of >15 days (69.0%; OR 1.12 (95% CI 0.94-1.33)): the live birth rate was also similar between groups (45.5% vs 51.5%, respectively; OR 1.14 (95% CI 0.97-1.35)). In all analyses, once a pregnancy was achieved, the length of the follicular phase or the length of elevated oestradiol >100 pg/ml no longer affected the pregnancy outcomes. LIMITATIONS, REASONS FOR CAUTION: The retrospective design of this study is subject to possible selection bias in regard to which patients at our clinic were recommended to undergo a natural FET compared to a fresh embryo transfer or programmed FET. To decrease the heterogeneity of our study population, we only included patients who had blastocyst embryo transfers; therefore, it is unknown whether similar results would be observed in patients with cleavage-stage embryo transfers. The retrospective nature of the study design did not allow randomized to a specific ovarian stimulation or ovulation trigger protocol. However, all patients were managed with the standardized protocols at a single center, which strengthens the external validity of our results when compared to a study that only evaluates one specific stimulation protocol. WIDER IMPLICATIONS OF THE FINDINGS: Our observations provide cycle-level characteristics that can be applied during a natural FET cycle to help optimize embryo transfer success rates. Physicians should consider the parameter of number of days that oestradiol is >100 pg/ml prior to the LH surge when determining whether to proceed with embryo transfer in a natural cycle. This cycle-specific characteristic may also help to provide an explanation for some failed transfer cycles. Importantly, our findings should not be used to determine whether to recommend a natural or a programmed FET cycle for a patient, but rather, to identify natural FET cycles that are not optimal to proceed with embryo transfer. STUDY FUNDING/COMPETING INTEREST(S): No financial support, funding, or services were obtained for this study. The authors do not report any potential conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Reproductive and obstetric outcomes in women of racial minorities aged 40 years and older undergoing IVF.

RESEARCH QUESTION: Do women of racial minorities aged 40 years or older have similar reproductive and obstetric outcomes as white women undergoing IVF? DESIGN: A retrospective cohort study conducted at a single academic university-affiliated centre. The study population included women aged 40 years or older undergoing their first IVF cycle with fresh cleavage-stage embryo transfer stratified by racial minority status: minority (black or Asian) versus white. Clinical intrauterine pregnancy and live birth rate were the primary outcomes. Preterm delivery (<37 weeks) and small for gestational age were the secondary outcomes. Odds ratios with 95% confidence intervals were estimated. P < 0.05 was considered to be statistically significant. RESULTS: A total of 2050 cycles in women over the age of 40 years were analysed, 561 (27.4%) of which were undertaken by minority women and 1489 (72.6%) by white women. Minority women were 30% less likely to achieve a pregnancy compared with their white (non-Hispanic) counterparts (adjusted OR 0.68, CI 0.54 to 0.87). Once pregnant, however, the odds of live birth were similar (adjusted OR 1.23, CI 0.91 to 1.67). Minority women were significantly more likely to have lower gestational ages at time of delivery (38.5 versus 39.2 weeks, P = 0.009) and were more likely to have extreme preterm birth delivery 24-28 weeks (5.5 versus 1.0%, P = 0.021). CONCLUSION: Minority women of advanced reproductive age are less likely to achieve a pregnancy compared with white (non-Hispanic) women. Once pregnancy is achieved, however, live birth rates are similar albeit with minority women experiencing higher rates of preterm delivery.

Reproductive and obstetric outcomes in mildly and significantly underweight women undergoing IVF.

RESEARCH QUESTION: What is the impact of low body mass index (BMI) on live birth rates and obstetric outcomes in infertile women treated with IVF and fresh embryo transfer? DESIGN: This was a retrospective cohort study of infertile patients in an academic hospital setting who underwent their first oocyte retrieval with planned autologous fresh embryo transfer between 1 January 2012 and 31 December 2018. The primary study outcome was live birth rate. Secondary outcomes were IVF treatment and delivery outcomes. Underweight patients were stratified into a significantly underweight group (body mass index [BMI] <17.5 kg/m2) and a mildly underweight group (BMI 17.5-18.49 kg/m2), and were compared with a normal-weight group (BMI 18.5-24.9 kg/m2). RESULTS: A total of 5229 patients were included (significantly underweight, 76; mildly underweight, 231; normal weight, 4922), resulting in 4798 embryo transfers. After oocyte retrieval, there were no significant differences between groups for total oocytes, mature oocyte yield and number of supernumerary blastocysts cryopreserved. Among women who had an embryo transfer, there were no significant differences in the live birth rates in significantly (31.0%, odds ratio [OR] 0.67, confidence interval [0.95, CI] 0.40-1.13) and mildly (37.7%, OR 0.95, CI 0.73-1.33) underweight patients compared with normal-weight patients (35.9%). Additionally, there were no statistically significant increased risks of preterm delivery, Caesarean delivery or a low birthweight (<2500 g) neonate. CONCLUSIONS: Mildly and significantly underweight infertile women have similar pregnancy and live birth rates to normal-weight patients after IVF treatment. In addition, underweight patients do not have an increased risk of preterm delivery (<37 weeks), Caesarean delivery or a low birthweight neonate.

Assessing the cognitive and behavioral development of 3-year-old children born from fathers with severe male infertility.

BACKGROUND: Despite the large number of babies born worldwide following intracytoplasmic sperm injection, concerns about the procedure's safety still exist owing to the use of suboptimal spermatozoa. Thus, follow-up of children conceived via intracytoplasmic sperm injection is highly recommended. We propose the use of parent-administered questionnaires to monitor the development of offspring conceived via intracytoplasmic sperm injection. OBJECTIVE: This study aimed to determine whether male infertility treatment affects offspring development. STUDY DESIGN: We compared obstetrical and neonatal outcomes and physical and psychological development of toddlers conceived via in vitro fertilization and intracytoplasmic sperm injection. Once newborns reached 3 years of age, participating patients were sent a set of parent-administered questionnaires, including the Ages and Stages Questionnaires; Prescreening Developmental Questionnaire 2; Peabody Developmental Motor Scales, Second Edition; Social Skills Rating System; Parenting Stress Index, Third Edition; and Child Behavior Checklist for Ages 2-3. Child development was measured by the Ages and Stages Questionnaires; Prescreening Developmental Questionnaire 2; and Peabody Developmental Motor Scales, Second Edition, questionnaires, whereas Social Skills Rating System; Parenting Stress Index, Third Edition; and Child Behavior Checklist for Ages 2-3 questionnaires were used to measure child behavior. The child's developmental or behavioral outcome was considered "abnormal" when he or she scored below average in ≥2 questionnaires from the respective category. We also conducted subanalyses to assess the effects of male genomic integrity, DNA fragmentation, chemical exposure, utilization of surgically retrieved spermatozoa, and extended embryo culture to determine the development of a child conceived via intracytoplasmic sperm injection. RESULTS: A total of 12,306 couples met the inclusion criteria for this study; 1914 of 7433 patients (25.8%) who underwent intracytoplasmic sperm injection and 451 of 4873 patients (9.3%) who underwent in vitro fertilization returned the questionnaires. Our comparison of obstetrical outcomes between the 2 groups did not reveal any significant differences in the mode of delivery distribution, with most mothers having uncomplicated vaginal deliveries. Furthermore, gender distribution, gestational ages, and birthweights were also comparable between children conceived via intracytoplasmic sperm injection and in vitro fertilization. However, children conceived via in vitro fertilization displayed impaired developmental characteristics compared with the intracytoplasmic sperm injection-conceived cohort (adjusted odds ratio, 0.72; 95% confidence interval, 0.5-0.9; P=.0004). There was no difference in child behavior. Furthermore, 3 cases of autism were reported, 1 case from the in vitro fertilization group and 2 from the intracytoplasmic sperm injection group, all conceived from couples with an older male partner. Ages and Stages Questionnaires outcomes were also compared for the offspring conceived via in vitro fertilization and intracytoplasmic sperm injection by gender; however, no significant differences were observed. In addition, 5 separate subanalyses were then conducted exclusively for the intracytoplasmic sperm injection-conceived group. Levels of spermatogenic failure, DNA fragmentation, and chemical exposure did not significantly affect offspring development. Interestingly, although the length of embryo culture did not seem to influence child development, the abnormal behavior rate was significantly higher in children from the day 3 embryo transfer cohort (adjusted odds ratio, 0.4; 95% confidence interval, 0.05-0.34; P=.04). Children conceived via intracytoplasmic sperm injection from ejaculated spermatozoa displayed impaired developmental and behavioral characteristics compared with toddlers conceived from surgically retrieved specimens (adjusted odds ratio, 4.9; 95% confidence interval, 1.2-20.7; P=.05). CONCLUSION: Most children conceived via intracytoplasmic sperm injection and in vitro fertilization are developing well without significant delays. Although the development of a child conceived via intracytoplasmic sperm injection was not affected by most of the variables assessed, those conceived from surgically retrieved spermatozoa were at a considerably lower risk of abnormal developmental and abnormal behavioral characteristics than offspring conceived from ejaculated specimens. However, given the small numbers of respondents available for many subgroups of interest, further studies of outcomes of children born from fathers with severe male factor infertility are warranted.

Tubal Ectopic Pregnancy with Undetectable Initial Serum ß-Human Chorionic Gonadotropin Level.

We present a case of a tubal ectopic pregnancy (EP) in a patient with an initially undetectable serum ß-human chorionic gonadotropin (ß-hCG) level. A 33-year-old woman in a same-sex relationship underwent timed donor intrauterine insemination. Her serum ß-hCG level was <5 mIU/mL 14 days after the intrauterine insemination. She reported menstrual bleeding 3 days after her negative pregnancy test and returned to the office 10 days later to begin a new treatment cycle. Her serum levels of estradiol, progesterone, and ß-hCG were 119 pg/mL, 6.1 ng/mL and 1157 mIU/mL, respectively. Transvaginal ultrasonography did not show an intrauterine pregnancy. Her ß-hCG level increased to 1420 mIU/mL the next day. She was diagnosed with a pregnancy of unknown location and treated with methotrexate. Her ß-hCG levels continued to increase despite 3 methotrexate doses, necessitating laparoscopy. The diagnostic laparoscopy demonstrated approximately 100 mL of hemoperitoneum in the posterior cul-de-sac with an intact right fallopian tube that was dilated at its distal end by the EP. A total right salpingectomy was performed. Her ß-hCG level was <5 mIU/mL 3 weeks later. The current case supports that although rare, an undetectable serum ß-hCG level does not completely rule out the diagnosis of an EP.