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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 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.

Study question Can whole exome sequencing (WES) of spermatozoal DNA provide insight into understanding the different steps that lead to inability of a couple to reproduce? Summary answer The identification of germline mutations can clarify different aspects of reproductive failure in couples with unexplained infertility. What is known already The limitation of a routine semen analysis in evaluating sperm characteristics, even according to the most stringent criteria, lies in its inability to provide substantial information on spermatozoa performance in ART. As a result, ancillary tests are being used to further assess the male gamete’s reproductive potential. More recently, WES of the male genome, carried out on somatic cells, has become a powerful technique that can potentially shed light on the genetic causes of infertility. Here, we aim to preferentially detect germline mutations by sequencing spermatozoal DNA to pinpoint genes related to different underlying etiologies for reproductive failure. Study design, size, duration In a 5-year period, 25 couples subdivided according to their ICSI outcomes were included in this study. Sperm aneuploidy assessment by fluorescent in situ hybridization (FISH) and copy number variant (CNV) analysis by WES were carried out on ejaculated specimens from consenting male partners. Following CNV analysis, gene mutation profiles were compared between the fertile (n = 10) and infertile cohorts (n = 15), as well as in relation to the reasons for reproductive failure. Participants/materials, setting, methods FISH was performed on at least 1,000 sperm cells with a threshold of > 1.6%. DNA was extracted and amplified from at least 500 spermatozoa (DNA concentration, 605±137 ng/ul; quality, 1.7±0.1 nm) for CNV analysis by WES. Mutations corresponding to the CNV were annotated and assessed using the CLC Genomic Server 9.0. Genes were considered duplicated or deleted when their read depth was >1.5 or Main results and the role of chance Couples (n = 25) (maternal age, 38.6±3yrs; paternal age, 39.7±5yrs) had normal somatic karyotypes with normal semen parameters (59.2±30x106/mL concentration, 44.8±18% motility) by WHO standards. The fertile (n = 10) cohort underwent 12 ICSI cycles, achieving an 82.6% (57/69) fertilization rate and 10/12 (83.3%) term pregnancies. The infertile cohort (n = 15) underwent 21 ICSI cycles, achieving a 66% (62/84) fertilization rate and 5/17 (29.4%) clinical pregnancies, all resulting in pregnancy loss. Sperm aneuploidy was consistently higher in the infertile (8.4%) versus fertile (4.0%) cohort (P For both cohorts, WES detected deletions responsible for sperm–egg fusion (ADAM3A) and acrosomal development (SPACA, SPATA), explaining the necessity for ICSI in these couples. The infertile cohort was characterized by 4 reasons for cycle failure: complete fertilization failure, poor embryo development, implantation failure, and pregnancy loss. Couples with complete fertilization failure (n = 4) had deletions (PLCZ1, PIWIL1) indicating a sperm-related oocyte-activating deficiency. Those with poor embryo development (n = 5) had mutations (HAUS1, KIF4A, XRN1) essential for centrosome integrity and spindle/microtubular stabilization. Couples who did not achieve pregnancy (n = 7) had a mutation (IL9R) in common related to cytokine constituents in the implantation pathway. Those with pregnancy losses (n = 5) had mutations (NLRP7, TP53) on post-implantation genes. Limitations, reasons for caution Several germline mutations, related to the different reasons for these couples’ reproductive failure, were identified. Although intriguing, these findings are still new and need to be validated in a larger study population. In addition, while maternal age was controlled for, we cannot definitively exclude other confounding female factors. Wider implications of the findings Additional screening methods for infertile couples, particularly those with unexplained infertility, can be used to clarify elusive factors underlying their reproductive ability. A genetic screening of spermatozoal DNA may therefore be considered a potential tool in precision medicine for the treatment of subtle male factor infertility. Trial registration number n/a

Electroejaculation has been successfully used for sperm procurement in anejaculatory men desiring fertility. However, electroejaculates typically have normal sperm numbers but poor motility, morphology, and functional deficiencies. Here we report the pregnancy outcome of a series of couples undergoing combined electroejaculation and in-vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI). In all, 13 couples underwent a total of 18 cycles. The aetiologies of anejaculation included history of retroperitoneal lymph node dissection for testicular cancers, spinal cord injury and psychogenic causes. ICSI was performed on 192 oocytes, resulting in a fertilization rate of 75.5%. A total of 15 embryo transfers were performed using a total of 51 embryos. Clinical pregnancy rate, as defined by positive fetal heart rate(s) using vaginal sonography, was 55.6% per retrieval; implantation rate was 33.3% per embryo. These rates appear to be similar to those obtained in standard IVF for non-male factor infertility, or ICSI for male factor infertility. The use of ICSI for electroejaculates undoubtedly provides these couples with the highest chance of pregnancy.