Is there a difference in endometrial immune cell expression at the time of embryo transfer in fresh vs. frozen transfer cycles? Our study is the first to compare the endometrial immune cell profile at the time of the embryo transfer between fresh and frozen cycles. Endometrial receptivity plays a crucial role in implantation success for both natural conception and assisted reproductive technology cycles; however, limited information is available about the endometrial environment during the actual implantation window. Although some data suggest a difference in pregnancy rates between fresh and frozen embryo transfers, the underlying mechanisms remain unclear. This study aims to address this gap by analyzing the endometrial milieu using an innovative approach that examines immune cells collected at the time of embryo transfer in both fresh and frozen transfer cycles. This study was approved by Institutional Review Board (# 49174). The study was designed as a prospective observational cohort study at a single academic fertility center. Fifty-two participants with embryos available for transfer were recruited, from which 42 underwent frozen embryo transfer (FET) and 10 underwent fresh embryo transfer (FrET). IVF protocols for FrET consisted of antagonist protocols with gonadotropin dose adjusted based on ovarian reserve, while FET cycles used hormone replacement protocols. All embryo transfers were performed under transabdominal ultrasound guidance. Upon completion of transfer and catheter verification, the transfer catheter tip was rinsed in IMDM medium containing 10% FBS. After centrifugation, pelleted cells were stained for the following surface markers: CD45, CD3, CD19, CD4, CD8, gamma delta TCR, CD25, CD127, CD56, CD14, and CD66b. The samples were acquired on Sony SP6800 Spectral Analyzer. Mann-Whitney U test was used to test statistical differences between groups. A statistically significant difference in the immune milieu was observed in patients undergoing FET compared to FrET cycles. In patients that received a FrET, there was a significantly higher expression of total immune cells (CD45+), 20.50%, compared to FET cycles, 6.60%, within all live cells (p = 0.0008). In addition, FrET cycles had an increase in the endometrial expression of total T cells (CD3+), 17.75%, compared to 2.70% T cells in the endometrium from patients undergoing FET cycles (p = 0.0051). Furthermore, patients undergoing FET cycles, had significantly greater endometrial expression of CD8+ T cells compared to FrET cycles, 20.5% vs. 2.30%, respectively (p = 0.0058). Finally, FET cycles were noted to have increased endometrial expression of B cells (CD19+) and NK cells (CD56+), while the endometrium for patients undergoing FrET cycles demonstrated greater GDT+ and T regulatory cells (CD4+CD25+CD127-), these differences did not reach statistical significance. There were similar endometrial expressions of T cells (CD4+), macrophages (CD14+), and neutrophils (CD66b+) among both types of cycles. Limitations of our study include the small sample size undergoing FrET. In addition, we are planning to compare the immune cell profiles between the two groups in relation to cycle outcomes. Furthermore, multicolor flow cytometry may provide additional immune cell composition of the immune milieu during the window of implantation. The endometrial milieu in general, and the immune environment in particular, differ between fresh and frozen cycles, and this could play a critical role in implantation. Identifying cycle specific immune cell profiles may reveal factors that optimize implantation, paving the way for diagnostic and therapeutic innovations to improve IVF outcomes. No

PROBLEM:Although endometrial receptivity is a key factor in influencing implantation in both naturally conceived and assisted reproductive technology (ART) cycles, very little is known about the endometrium milieu around the time of implantation. Previous studies have demonstrated the presence of several cytokines in the endometrium that affect implantation. However, there is lacking data about the presence of immune cell subtypes within the endometrium and in the uterine cavity at the time of implantation. METHOD OF STUDY:This study was approved by the Institutional Review Board (# 225589). The study was designed as a prospective observational cohort study between May 2021 and December 2022 at a single academic-based fertility center. All patients underwent at least one In Vitro Fertilization (IVF) cycle and have frozen embryos. Twenty-four participants were recruited for this study which was conducted during the frozen embryo transfer (FET) cycle regardless of the outcome of previous cycles. Two samples were acquired from each subject, denoted as lower and upper. A trial transfer catheter was introduced under ultrasound guidance into the lower uterine segment. Upon removal, the tip was rinsed in IMDM medium containing 10% FBS (lower uterus). A transfer catheter was then loaded with the embryo that was placed in the upper uterus under ultrasound guidance. The tip of the transfer catheter was rinsed in separate aliquot of the above media (upper uterus). After centrifugation, pelleted cells were stained for the following surface markers: CD45, CD3, CD19, CD4, CD8, gamma delta TCR, CD25, CD127, CD66b, CD14, CD16, CD56 and acquired on Sony SP6800 Spectral Analyzer. RESULTS:Upon staining the pelleted cells, we were able to identify viable leukocytes from samples obtained from both, upper and lower uterus (0.125 × 106 cells ± SD 0.32), (0.123 × 106 cells ± SD 0.12), respectively. Among total viable cells, there was no significant difference in both percent and number of CD45+ cells between the upper and lower uterus (9.88% ± 6.98 SD, 13.67% ± 9.79 SD, p = .198) respectively. However, there was significantly higher expression of CD3+ (p = .006), CD19+ (p = .032) and CD14+ (p = .019) cells in samples collected from upper compared to lower uterus. Within all CD3+ cells, we found that gamma delta T cells (GDT) were the major population of T cells in both upper and lower uterus. In contrast, CD8+ T cells were significantly higher in the lower uterus when compared to the upper uterus (p = .009). There was no statistically significant difference in the expression of CD4+ T cells, T regulatory cells (CD4+CD25+CD127-), NK cells (CD56+), neutrophils (CD66b+) and FcγRIII+ cells (CD16+) between upper and lower uterus. CONCLUSIONS:We believe the immune milieu at the time of embryo transfer will affect implantation. Understanding the composition of immune cells will guide further research in identifying optimal immune milieus that favor implantation. Comprehensive analysis of endometrium is expected to lead to new diagnostic and therapeutic approaches to improve IVF outcomes.

Abstract Study question Is there an association between live birth and uterine cavity immune cell subsets collected at the time of frozen embryo transfer (FET)? Summary answer A positive correlation exists between live birth and number of uterine cavity immune cells, as well as T regulatory cells at the time of FET. What is known already Several strategies have been deployed to improve pregnancy rate during IVF, with little success. Although selecting the euploid embryo was thought to improve pregnancy rate, implantation rate of 50-60% has been reported. Endometrial receptivity plays a critical role in the success of implantation and could be the missing link in improving pregnancy rate. While certain cytokines within the endometrium are recognized for influencing implantation, information on uterine milieu at time of implantation is limited. Immune cell profile at the time of FET may reflect on endometrial receptivity and could be utilized as a marker for predicting implantation and successful pregnancy. Study design, size, duration Institutional Review Board approval was obtained for this study (#49174). This prospective observational cohort study was conducted at a single academic-based fertility clinic. A total of 24 subjects were recruited between May 2021 through December 2022. Eligible participants were those who had undergone at least one cycle of IVF with at least one frozen embryo available for FET. Live birth was defined as birth of one of more live-born infants. Participants/materials, setting, methods Under ultrasound guidance, trial transfer catheter was placed in lower uterine segment, then rinsed in IMDN medium with 10% FBS (lower). Embryo was placed in the upper uterus and the transfer catheter tip was rinsed in separate sample of above media (upper). Collected cells were centrifuged and stained for the following surface markers: CD45, CD3, CD19, CD4, CD8, gamma delta TCR, CD25, CD127, CD66b, CD14, CD16, CD56. Statistical analysis was performed with Pearson correlation test. Main results and the role of chance All participants underwent elective single embryo transfer under ultrasound guidance. Out of the 24 patients participating in the study, 13 patients had a live birth (54%), one patient had a biochemical pregnancy (4%), 10 patients did not achieve pregnancy (42%), and there were no miscarriages. When patients with a live birth were compared to those without a live birth, there was no significant difference in their age (P 0.87), body mass index (P 0.64) or ethnicity (P 0.94). There was no association between the relative expression and subtypes of different immune cells isolated from the lower uterine segment and pregnancy outcome. However, in cells isolated from upper uterus, there was a positive correlation between live birth and total immune cells (CD45+; P 0.04) and T regulatory cells (P 0.03) at the time of FET. There was no statistically significant association with live birth noted with the remainder of the analyzed cells, including neutrophils (CD66b), NK cells (CD56), T cells (CD3) and other subsets (GDT, CD8, CD4), B cells (CD19), monocytes (CD14), and Fc receptor positive cells (CD16). Limitations, reasons for caution Our study’s limitations include the number of participants and the range of flow cytometry parameters that were analyzed. With the use of multicolor flow cytometry, additional detailed data can be obtained on immune cell diversity and expression. Wider implications of the findings We posit that the uterine milieu during embryo transfer could influence implantation. Analyzing immune cell association with live birth can direct future studies toward identifying conditions conducive to implantation, paving the way for novel diagnostics and treatments to enhance implantation and subsequently live birth rate. Trial registration number not applicable

To investigate whether day of embryo biopsy and embryo quality has a direct effect on the incidence of low DNA and inconclusive results on PGT reports. Retrospective analysis of patients undergoing PGT-A cycles (Oct. 2019 and March 2023). Embryos were cultured in benchtop incubators with single step culture media until reaching blastocyst stage, when trophectoderm biopsy was performed (either day 5 or 6). SART embryo morphology grading criteria were used, and embryos were classified into three groups (good, fair, and poor). Chi-square was used for statistical analysis and p< 0.05 considered significant. A total of 1,294 embryos were biopsied, 853(65.9%) on day 5 and 441 (34.1%) on day 6. The rate of euploid embryos was higher on day 5 (470/853, 55.1%) than day 6 (161/441, 36.5%) (p< 0.001). Aneuploidy rates were lower on day 5 embryos (299/853, 35%) than on day 6(210/441, 47.7%) (p<0.008). Embryo mosaicism on day 5 was 2.46% (21/853) vs 2.72% (12/441) on day 6. The incidence of low DNA among day 5 embryos was 4.45% (38/853) vs 8.84 (39/441) on Day 6 (p<0.003). The incidence of inconclusive result was 2.93% (25/853) vs 4.31% (19/441) on day 5 and 6, respectively (p=0.211) (table).Tabled 1Table 1. Comparison between low DNA on day 5 and day 6 according to GradesGradesDay 5Day 6P-valueN%N%Good26/5354.88%11/10610.38%0.039*Fair12/2724.41%21/2179.68%0.032*Poor0/460.0%7/1185.93%0.101 Open table in a new tab There were 535 good, 272 fair, and 46 poor embryos on day 5 of development and the incidence of low DNA was 4.84% (26/535), 4.41 (12/272), and 0% (0/47), respectively. There were 106 good, 217 fair, and 118 poor embryos on day 6 and the incidence of low DNA results was significantly higher than the corresponding day 5 embryos 10.38% (11/106), 9.68% (21/217), and 5.93% (7/118) (p= 0.039) and (p=0.032) for good and fair morphology, respectively. There was no significant difference in the incidence of low DNA in poor embryos between day 5 and day 6, although it was more frequent on day 6 (p 0.101). The incidence of inconclusive results according to good, fair, and poor morphology grading (on day 5) were 1.68% (9/535), 5.15% (14/272) and 4.35% (2/14), respectively; while on day 6 were 3.77% (4/106), 2.76% (6/217), and 7.63% (9/118), respectively, (p=NS). Embryo grade and the day of biopsy showed a direct correlation on the incidence of low DNA and inconclusive results after PGT-A. It is plausible that embryos with a slower growth rate (day 6 blastocysts) and of poorer morphological grading are trying to eliminate apoptotic cells that could be biopsied, leading to a higher incidence of no actionable results.

Since the introduction of assisted reproductive technology, increased proportion of women have delayed childbearing for a variety of reasons. The current literature on fertility preservation in ethnic minorities does not assess the frequency and usage of oocyte cryopreservation (OC) among this group. The purpose of this study is to determine whether ethnic minorities are seeking OC services at the same rate as compared to their non-Hispanic White counterparts. Demographic information and OC outcomes were collected for all patients presenting for OC between 01/01/2020 to 12/31/2022 at a single fertility center located in South Florida. Descriptive statistics (T-test and Chi square test) were used to assess the data. A total of 79 patients presented for OC however 3 did not provide race and/or ethnicity and were excluded from the study. Minority groups (Hispanic, Black, or Asian) were the majority, representing 51 (67%) of patients undergoing OC vs. 25 (33%) non-Hispanic White. Thirty-eight patients were Hispanic (50%), representing the largest proportion of ethnic minorities. There was no difference in age amongst minorities and non-Hispanic white patients, 31.2 +/- 5.8 vs. 33.2 +/- 5.9, p=0.17. Furthermore, there was no difference in the indications for OC in minorities compared to White non-Hispanic patients (p=0.62). The main indication for OC was elective in 44 patients (57.9%) of which 28 patients (64%) were ethnic minorities. Fertility preservation in the setting of new cancer diagnosis made up 29 patients (38.2%) of which 20 patients (69%) were ethnic minorities. Two transgender males (2.6%) participated in OC, both ethnic minorities, and one patient (1.3%) performed OC prior to myomectomy. Additionally, there was no significant difference in the mean number of oocytes retrieved between ethnic minorities and non-Hispanic White patients, 14.7 +/- 10.2 vs. 16.2 +/- 9.1, p=0.50, respectively. Our data shows that there is no disparity in access and utilization of OC services, whether for elective or oncofertility indications, in a geographic location with predominance of ethnic minorities.

To assess the blastocyst rate as indirect measure of both patient's gamete competence and laboratory performance before and during the COVID pandemic years.

The finding of conjoined oocytes is a rare occurrence that accounts for only 0.3% of all human retrieved oocytes. This phenomenon is quite different from that of a traditional single oocyte emanating from one follicle, and may result in dizygotic twins and mosaicism. Given the insufficient evidence on how to approach conjoined oocytes, their fate is variable among different in vitro fertilization (IVF) centres. In this observational report, we propose a new protocol for the use of these conjoined oocytes using intracytoplasmic sperm injection (ICSI), laser-cutting technique and next-generation sequencing (NGS). The first case report demonstrates that conjoined oocytes can penetrate their shared zona pellucida (ZP) at Day 6. The second case is that of a 25-year-old female patient who underwent a successful embryo transfer cycle after removal of one oocyte in which a pair of conjoined human oocytes underwent ICSI, laser-cutting separation and NGS testing. The patient achieved pregnancy and gave birth to single healthy female originally derived from conjoined oocytes. This case provided a means through which normal pregnancy may be achieved from conjoined oocytes using laser-cutting separation techniques. The protocol described may be especially beneficial to patients with a limited number of oocytes.

Preimplantation genetic testing for aneuploidy (PGT-A) is increasingly used in patients undergoing in vitro fertilization (IVF).1 However, this is not without debate.2 Physicians must also factor in additional costs for aneuploidy testing. Neal et al. created a decision analytic model showing IVF with PGT-A can reduce costs when compared to IVF alone in patients with more than 1 embryo.1 However, the multi-center STAR trial by Munne et al. concluded PGT-A did not improve overall pregnancy outcomes in all women.2 With this new finding, we question the cost efficacy of PGT-A. To our knowledge, no cost efficacy studies have been done with the findings of the STAR trial. We plan to demonstrate a theoretical model using the new findings from the STAR trial in which we will determine if PGT-A is cost effective. An IRB exempt hypothetical cost-analysis study. In this hypothetical study, we created a comparison model with 100 patients undergoing single frozen embryo transfer in a PGT-A group and 100 control IVF patients. We performed an analysis between patients less than 35 years old and a subsequent analysis looking at patients 35-40 years old. Procedural costs were based on those used in our clinic. All costs up to the point of embryo biopsy were assumed to be equal. Pregnancy and live birth rates in the non-PGT group were based on SART data. The percentage of euploid embryos in the PGT-A group were obtained from the STAR trial. Each group of patients was then taken thru 2 frozen embryo transfer cycles. After this, the cost per live birth in each cycle and the average cost per live birth over two cycles was determined. In patients less than 35 years old undergoing IVF without PGT, the cost per live birth in the 1st cycle was $12,500, while the cost per live birth after the second cycle was $26,785. In women under 35 undergoing IVF with PGT-A, the cost per live birth after one cycle was $21,250, and the cost after two cycles was $31,250. In patients 35-40 years old using IVF without PGT-A, the cost per live birth was $18,518 after one cycle and $42,800 after the second cycle. In the 35-40 year old PGT-A group, the cost per live birth after one cycle was $26,176. After two cycles, the cost was $36,176. The average cost per live birth over 2 cycles in patients under 35 with and without PGT-A was $24,583 and $17,417, respectively. The average cost per live birth over 2 cycles in patients 35-40 years old with and without PGT-A was $29,637 and $27,190, respectively. This data further demonstrates PGT-A is not cost effective in patients under 35 years old. In light of the new data from the STAR trial, in patients 35-40 years old, PGT-A is cost neutral if a patient requires two cycles of frozen embryo transfer.

Introduction: Polycystic ovarian syndrome (PCOS) is a common cause of female infertility. Clomiphene citrate (CC) is a first line treatment for infertility secondary to PCOS, in addition to Letrozole. After unsuccessful cycles using CC and timed intercourse, pregnancy may be achieved using in-vitro fertilization (IVF), bypassing gonadotropins. We explore the most efficient and cost-effective way to obtain at least 70% live-birth rate in PCOS patients who fail first-line treatment. Materials and methods: A review of relevant trials using PUBMED was performed to obtain pregnancy rates of women with PCOS undergoing various treatments. Six randomized trials were included in this study. We used 761 cycles from these trials. Four different protocols were structured: protocol 1 consisted of 3 cycles of CC/intrauterine insemination (IUI) followed by 3 cycles of human menopausal gonadotropin stimulation with IUI followed by 2 cycles of IVF/intracytoplasmic sperm injection (ICSI). Protocol 2 involved 3 cycles of CC/IUI followed by 2 cycles of IVF/ICSI. Protocol 3 was comprised of 3 cycles of human menopausal gonadotropin/IUI followed by 2 cycles of IVF/ICSI, and protocol 4 was composed of 2 cycles of IVF/ICSI and 1 cycle of frozen embryo transfer. Each protocol was analyzed for cost per live birth and time to live birth. Results: Protocol 1 yielded a live-birth rate of 75%, costing $34,923 per live-birth achieved over a 10-month period. Protocol 2 yielded a pregnancy rate of 71%, costing $32,172 over 7 months. Protocol 3 yielded a pregnancy rate of 73%, costing $39,812 over 7 months. Lastly, protocol 4 yielded a pregnancy rate of 70%, costing $37,884 per pregnancy over a 5-month period. Conclusion: Protocol 4 was the most efficient, reaching a 70% live birth rate in a 5-month period. Protocol 2 was the most cost-effective, with a total cost of $32,172 per live birth. These results will assist physicians in counseling PCOS patients with subfertility to determine the optimal treatment method.

Background: In vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI) has been increasingly used to treat an expanding subset of infertile patients; However, IVF/ICSI is not without inherent risks. The goal of this study was to determine if pre-wash and/or post-wash semen parameters are useful in determining the best method of fertilization between IVF and ICF/ICSI. Methods and Findings: A retrospective chart review was performed on 211 cycles performed at the UHealth Reproductive and Fertility Center at the University of Miami Hospital between the years of 2008-2014. 126 of those cycles were performed with ICSI and the remaining 85 were performed with conventional IVF. The volume, concentration, and motility for each semen sample were measured. These semen parameters were used to calculate total motile count (TMC), volume (ml), concentration (million/ml), and motility (percentage). This was done in both prewash and post-wash samples. We compared patient characteristics, pre/post-wash parameters, and outcomes for IVF with conventional fertilization vs. IVF/ICSI. The main outcome studied was fertilization rate. A receiver operating characteristic (ROC) curve analysis determined that a post-wash threshold of 25 million TMC achieved a 70% fertilization rate in conventional IVF patients. This rate is comparable to that achieved with ICSI. Conclusion: There is evidence to show that pre-wash and post-wash parameters of total motility count of sperm can be used to make decisions regarding use of conventional fertilization versus ICSI in patients undergoing IVF.

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology1 Apr 2016MP70-09 INDICATORS OF INFLAMMASOME ACTIVATION IN MEN WITH ABNORMAL SEMEN PARAMETERS Emad Ibrahim, Karen Ibrahim, Michael Jurewicz, Teodoro Aballa, George Attia, Charles Lynne, and Nancy Brackett Emad IbrahimEmad Ibrahim More articles by this author , Karen IbrahimKaren Ibrahim More articles by this author , Michael JurewiczMichael Jurewicz More articles by this author , Teodoro AballaTeodoro Aballa More articles by this author , George AttiaGeorge Attia More articles by this author , Charles LynneCharles Lynne More articles by this author , and Nancy BrackettNancy Brackett More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1434AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Assembly of the inflammasome complex results in caspase-1 activation which promotes the maturation and secretion of the pro-inflammatory cytokines IL-1β and IL-18. Evidence of inflammasome activation and the consequent elevation of semen cytokines has been reported as a possible cause of abnormal semen quality in men with spinal cord injury (SCI) [Fertil Steril 2013;99:118-24, Hum Reprod 2014;29:2368-73]. This study sought to determine if indicators of inflammasome activation were evident in non-SCI men with abnormal semen parameters. METHODS Semen was obtained from men with abnormal semen parameters who were being evaluated in an infertility clinic (n=131). Each of these men was assigned to a group depending on his semen parameters. Oligo: men with low sperm concentration but normal sperm motility (n=29); Astheno: men with low sperm motility (<40%) but normal sperm concentration (≥20 million/cc) (n=14); Oligo+Astheno: men with combined low sperm concentration and low sperm motility (n=70); Terato: men with normal sperm count, normal sperm motility, but low sperm morphology (<4%) (n=8); Azo: men with no sperm in their ejaculate (n=10). Over 80% of the men in the Oligo, Astheno and Oligo+Astheno groups also had low sperm morphology. Semen from healthy normospermic men (n=10) served as control material. Seminal plasma was separated from semen by centrifugation and analyzed for caspase-1, IL-18, and IL-1β concentrations using ELISA (R&D Systems, Minneapolis, MN). Comparisons between controls and patient groups were made by student's t-test. Statistical significance was considered at p ≤ 0.05. RESULTS Caspase-1 concentrations were significantly elevated in all groups compared to controls. IL-1β concentrations were elevated in all groups compared to controls but reached statistical significance only in the Oligo+Astheno, and the Terato groups. IL-18 concentrations were also elevated in all groups compared to controls but reached statistical significance only in the Oligo+Astheno group. (Table 1). CONCLUSIONS This study shows elevation of inflammasome activation indicators (caspase-1, IL-1β and IL-18) in semen of patients presenting for an infertility workup. Further investigation of the inflammasome in various etiologies of male infertility may result in therapeutic interventions. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e909 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Emad Ibrahim More articles by this author Karen Ibrahim More articles by this author Michael Jurewicz More articles by this author Teodoro Aballa More articles by this author George Attia More articles by this author Charles Lynne More articles by this author Nancy Brackett More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...