The role of endometrial staining for CD138 as a marker of chronic endometritis in predicting live birth.

PURPOSE: Chronic endometritis (CE) is diagnosed via endometrial biopsy and staining for plasma cells. A threshold plasma cell count that identifies CE and predicts pregnancy outcomes has not been established, and the prevalence of plasma cells in the general infertile population is unknown. The purpose of this study was to determine the prevalence of plasma cells in the general infertile population and whether a threshold exists which predicts live birth. METHODS: Endometrial samples were obtained prospectively from 80 women undergoing IVF, embedded in paraffin, and stained for plasma cells using mouse mono-clonal antibody for CD138. Slides were reviewed at 20× magnification and 10 random images captured. Three reviewers graded each image for plasma cells. Participants underwent single, euploid, and frozen blastocyst transfer. RESULTS: Forty-nine percent of samples had ≥1 plasma cell across 10 HPFs, 11% had ≥5 cells across 10 HPFs, and 4% had ≥10 cells across 10 HPFs. There was no difference in prevalence between those who did and did not achieve live birth. Using thresholds of 1, 5, and 10 plasma cells per 10 HPFs, there were no differences in implantation, clinical pregnancy, clinical pregnancy loss, or live birth rates between patients with and without CE. CONCLUSION: Endometrial plasma cells are present in half the general infertile population and do not predict implantation, clinical pregnancy, clinical pregnancy loss, or live birth rates at low levels.

Varying levels of serum estradiol do not alter the timing of the early endometrial secretory transformation.

STUDY QUESTION: Do supraphysiologic estradiol (E2) levels in the ranges attained during normal and high response superovulation cycles modify the onset of endometrial secretory transformation? SUMMARY ANSWER: Highly supraphysiologic levels of E2 do not alter the ability of physiologic levels of progesterone (P4) to induce secretory transformation. WHAT IS KNOWN ALREADY: Previous studies have demonstrated that premature P4 elevations during IVF cycles are associated with a decrement in clinical pregnancy rates after fresh embryo transfer due to shifts in the window of implantation (WOI). However, alterations in the onset of secretory transformation may not apply uniformly to all patients. High responders with supraphysiologic E2 levels accompanied by similar subtle increases in P4 have not been shown to have decreased sustained implantation rates. This prospective investigation in which whole-genome transcriptomic and methylomic analysis of the endometrium is performed for individual patients under a range of E2 concentrations brings clarity to a long-debated issue. STUDY DESIGN, SIZE, DURATION: A randomized, prospective and paired trial was conducted in which 10 participants were enrolled and randomized to the order in which they completed three distinct uterine stimulation cycles, each at a specific E2 concentration: physiologic (∼180 pg/ml), moderately supraphysiologic (600-800 pg/ml) or supraphysiologic (2000 pg/ml). Target E2 ranges were selected to mimic those seen in natural, controlled ovarian stimulation and IVF cycles. E2 valerate was administered in order to maintain stable E2 levels for 12 days followed by intramuscular P4 in oil 10 mg/day for two doses, after which an endometrial biopsy was performed. A total of 30 endometrial biopsies were included in a whole-genome transcriptomic and methylomic analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Healthy volunteers without a history of infertility were included in this study at a single large infertility center. DNA was isolated from the endometrial biopsy specimens and bisulfite sequencing was performed to construct a methylation array. Differential methylation analysis was conducted based on differences in M-values of individuals across treatment groups for each probe as well as carrying out t-tests. RNA was isolated for RNA-Seq analysis and gene expression values were compared using DESeq2. All analyses were performed in a pairwise fashion to compare among the three stimulation cycles within individuals and secondarily to compare all participants in each of the cycles. MAIN RESULTS AND THE ROLE OF CHANCE: The mean peak E2 and P4 levels were 275 pg/ml and 4.17 ng/ml in the physiologic group, 910 pg/ml and 2.69 ng/ml in the moderate group was, and 2043 pg/ml and 2.64 ng/ml in the supraphysiologic group, respectively. Principal component analysis of 834 913 CpG sites was performed on M-values of individuals within the low, moderate and supraphysiologic conditions in a paired approach. There were no differences in genome-wide methylation within participants across E2 groups. A paired analysis revealed that gene expression profiles did not differ within the same individual at each of the three E2 levels. No significant alterations in gene expression as related to endometrial physiology were identified between the low, moderate and supraphysiologic groups in an inter-participant analysis. LIMITATIONS, REASONS FOR CAUTION: Although each participant completed a physiologic cycle in which E2 levels were maintained in a range that would simulate a natural cycle, our findings are limited by lack of an unmedicated control to assess if there was a potential effect from E2V. Additionally, our results were obtained in fertile individuals, who may have a different endometrial response compared to an infertile population. Despite the whole genomic endometrial assessment and rigorous, paired study design, the sample size was limited. WIDER IMPLICATIONS OF THE FINDINGS: Given that the endometrial response to P4 is unaffected by E2 levels in the supraphysiologic range, diminutions in implantation seen in stimulated cycles may result from embryonic-endometrial dyssynchrony following early P4 elevations or slowly blastulating embryos, which occur independently of the magnitude of the E2 rise. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Foundation for Embryonic Competence, Basking Ridge, NJ, USA. Dr E.S. reports consultancy work for The Foundation for Embryonic Competence, Basking Ridge, NJ, USA. The other authors declare no conflict of interests related to this topic. TRIAL REGISTRATION NUMBER: NCT02458404.

Worth the wait? Day 7 blastocysts have lower euploidy rates but similar sustained implantation rates as Day 5 and Day 6 blastocysts.

STUDY QUESTION: Does the reproductive potential of embryos change when blastocyst development takes longer than the traditionally accepted 5 days when accounting for aneuploidy and endometrial-embryo asynchrony? SUMMARY ANSWER: Aneuploidy increases with increasing duration of blastulation, but if blastocyst morphologic quality and endometrial-embryo asynchrony are controlled for, euploid Day 7 embryos have similar sustained implantation as compared to Days 5 and 6 euploid blastocysts. WHAT IS KNOWN ALREADY: The relative contributions of diminished embryo quality versus endometrial and embryo asynchrony to poor outcomes associated with embryos cultured past Day 6 are not clear. Asynchrony can be eliminated by embryo vitrification with transfer in a subsequent month after retrieval. STUDY DESIGN, SIZE, DURATION: Retrospective cohort study of patients from a single center attempting conception through ICSI and utilizing preimplantation genetic testing for aneuploidy screening (PGT-A) from January 2017 to September 2018. Cycles were excluded if they utilized surgical sperm or preimplantation genetic testing for monogenetic/single gene defects. ICSI cycle outcomes from 2586 patients were evaluated for ploidy status of embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS: Only patients undergoing single, euploid frozen embryo transfer were included when analyzing cycle outcomes by day of blastocyst expansion of the transferred embryo (n = 2130). Ploidy rates by the day upon which an embryo was considered to be usable (denoted, 'usable blastulation day') were determined so as to assess the contribution of aneuploidy to slow embryo development. Outcomes of euploid frozen single embryo transfers (SET) of Day 7 embryos were evaluated to assess the reproductive potential associated with embryos that were slowly developing for reasons other than aneuploidy. Analyses were adjusted by maternal age and blastocyst morphology. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, 67.7% (n = 3508) of usable Day 5 blastocysts were euploid, 52.1% (n = 5560) of usable Day 6 blastocysts were euploid and 43.1% (n = 229) of usable Day 7 embryos were euploid (Day 5 versus Day 6: odds ratio (OR) 0.7 (95% CI, 0.64-0.76), P < 0.001; Day 5 versus Day 7: OR 0.56 (95% CI, 0.46-0.69), P < 0.001; Day 6 versus Day 7: OR 0.81 (95% CI, 0.67-0.99), P = 0.036). Stratified by Society for Assisted Reproductive Technology maternal age groups, a reduction in the prevalence of euploidy by increasing time to embryo blastulation was still seen. The sustained implantation rate (SIR) was similar after euploid SET of Days 5 and 6 embryos (overall, 68.9% (95% CI, 66.0-71.6) and 66.8% (95% CI, 63.8-69.7), respectively; P = 0.81). SIR after euploid Day 7 SET appeared slightly lower than that of Days 5 and 6 embryos (52.6% (95% CI, 35.8-69.0); (Day 5 versus Day 7: OR, 0.67 (95% CI, 0.32-1.41), P = 0.29; Day 6 versus Day 7: OR 0.58 (95% CI, 0.28-1.2), P = 0.14)) but did not achieve statistical significance. LIMITATIONS, REASONS FOR CAUTION: The primary limitation is the low number of Day 7 blastocyst transfers that limits statistical power. Additionally, the retrospective nature of this study may prevent full elucidation of potential biases with respect to culture, morphologic assessment and selection of Day 7 embryos for transfer. WIDER IMPLICATIONS OF THE FINDINGS: Routine culture through Day 7 may successfully increase the pool of transferrable embryos for patients who would otherwise have no usable embryos if culture terminated on Day 6. This is particularly true for older patients (i.e. greater than 35 years of age), whose embryos take longer to blastulate and, therefore, are more susceptible to cycle cancelation. Additionally, as evidenced by an adequate overall SIR of 52.6% after euploid SET of Day 7 blastocysts, embryos developing to a usable blastocyst on Day 7 are likely within the 'window of blastulation.' STUDY FUNDING/COMPETING INTEREST(S): None.

When next-generation sequencing-based preimplantation genetic testing for aneuploidy (PGT-A) yields an inconclusive report: diagnostic results and clinical outcomes after re biopsy.

PURPOSE: To describe diagnostic results following re-biopsy of blastocysts with inconclusive results on preimplantation genetic screening for aneuploidy (PGT-A) and to evaluate the reproductive potential of re-biopsied blastocysts. METHODS: This retrospective cohort study included all trophectoderm biopsies submitted for PGT-A by a large in vitro fertilization center to a single genetics laboratory from June 2016 to October 2018. PGT-A was performed using next-generation sequencing (NGS). No-result blastocysts that underwent re-biopsy were subsequently classified as euploid, aneuploid, mosaic/segmental, or no-result. Ongoing pregnancy and clinical loss rates were assessed following transfer of re-biopsied blastocysts. Logistic regressions were conducted to account for age and blastocyst morphology. RESULTS: Of the trophectoderm biopsies submitted for PGT-A, 635/25,199 (2.5%) were categorized as no-result. Those that underwent re-biopsy (n = 250) had a 95.2% diagnostic rate with 140 (56.0%) receiving euploid diagnoses. Thirty-six re-biopsied blastocysts deemed euploid were subsequently transferred, resulting in 18 (50.0%) ongoing pregnancies and 5 (13.9%) clinical losses. After adjusting for age and blastocyst morphology, there remained a lower ongoing pregnancy rate and a trend towards higher clinical loss rate following transfer of a re-biopsied blastocyst. When compared to blastocysts that underwent the same number of vitrification-warming cycles but only one biopsy, there were no differences in outcomes. CONCLUSIONS: Failure to obtain an analytical result does not change the probability that a given blastocyst is euploid. Pregnancy outcomes following transfer of re-biopsied blastocysts are favorable, but further data must be accrued for an adequately powered comparison with outcomes after transfer of blastocysts biopsied once.

Preclinical validation of a targeted next generation sequencing-based comprehensive chromosome screening methodology in human blastocysts.

STUDY QUESTION: Can a novel targeted next generation sequencing (tNGS) platform accurately detect whole chromosome aneuploidy in a trophectoderm biopsy and provide additional information to improve testing? SUMMARY ANSWER: Karyotypes obtained by tNGS were concordant with other validated platforms and single nucleotide polymorphism genotyping information obtained can be used for improved detection and quality control. WHAT IS KNOWN ALREADY: qPCR-based whole chromosome aneuploidy screening is highly accurate in comparison to other common methods and has been shown to improve IVF success in two randomized clinical trials. With aneuploidy screening becoming standard of care in many IVF centres, there is a need to develop platforms with high throughput, low cost capabilities. STUDY DESIGN SIZE, DURATION: Twelve well-characterized cell lines were obtained from a commercial cell line repository and 31 discarded human blastocysts were obtained from 17 IVF patients who underwent comprehensive chromosome screening (CCS). PARTICIPANTS/MATERIAL, SETTING, METHODS: All samples were processed using a unique amplification strategy which directly incorporated sequencing library adapters and barcodes. Sequencing was performed on an Ion Torrent Proton. A custom bioinformatics pipeline was used to determine the karyotype for each sample. The consistency of tNGS diagnoses with either conventional karyotyping of cell lines or quantitative real-time PCR based CCS of blastocyst biopsies was evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: Overall consistency per sample of tNGS based CCS in 5-cell samples from a variety of cell lines was 99.2%. In the blinded analysis of rebiopsies of aneuploid blastocysts, an overall targeted tNGS CCS consistency of 98.7% was observed per sample. These data demonstrate the ability of tNGS based CCS to provide an accurate and high throughput evaluation of aneuploidy in the human blastocyst. LARGE SCALE DATA: Not applicable. LIMITATIONS REASONS FOR CAUTION: This study is limited to whole chromosome aneuploidy, as mosaicism and segmental aneuploidy have not been investigated. WIDER IMPLICATIONS OF THE FINDINGS: These data show an accurate, high throughput method, and with the greater depth of each amplicon sequenced in comparison to commercial kits, there is greater application available for single nucleotide polymorphism based analysis for quality control. STUDY FUNDING/COMPETING INTERESTS: This study was funded through intramural research funds provided by the Foundation for Embryonic Competence. There are no competing interests.

Diminished ovarian reserve and poor response to stimulation in patients <38 years old: a quantitative but not qualitative reduction in performance.

STUDY QUESTION: Do infertile women aged <38 years with quantitative evidence of diminished ovarian reserve and/or poor response to stimulation also exhibit poor oocyte quality as measured by blastulation rates, aneuploidy rates, and live birth rates? SUMMARY ANSWER: Young women with evidence of accelerated follicular depletion, either by precycle ovarian reserve testing or postcycle evidence of low oocyte yield, exhibit equivalent blastulation rates, aneuploidy rates and live birth rates per euploid embryo transfer as age-matched controls with normal precycle and postcycle parameters. WHAT IS KNOWN ALREADY: Previous studies are conflicted as to whether women with evidence of diminished ovarian reserve and/or poor ovarian response are also at increased risk of exhibiting evidence of poor oocyte quality. Most prior studies have failed to adequately control for the confounding effect of female age on typical markers of oocyte quality in poor responders. The rate of follicular depletion occurs at around 38 years on average; thus, evidence of quantitative depletion before this would indicate a premature diminution of ovarian reserve and allow evaluation of whether markers of oocyte quality are tied to quantitative markers. STUDY DESIGN, SIZE, DURATION: This was a retrospective cohort study at a single center between 2012 and 2016. This time frame was specifically chosen as all embryos were cultured to the blastocyst stage at this center during the study period (no cleavage stage transfers were performed). Two comparisons were made: precycle assessment of ovarian reserve (based on anti-mullerian hormone (AMH) level) and postcycle oocyte yield results. For each comparison, patients in <10th percentile were compared to patients in the interquartile range (IQR) with respect to blastulation rate, aneuploidy rate and live birth rate. A mixed effects model was created to control for female age (in the <38 year old range) and correlation among oocytes from a given cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS: For the precycle blastulation analysis, only patients with AMH data available were included (345 patients with AMH in the <10th percentile versus 1758 patients with AMH in the 25th to 75th percentile (IQR)). To compare aneuploidy rates, the subset of these patients who pursued preimplantation genetic testing for aneuploidy (PGT-A) was then analyzed (124 patients in the <10th percentile versus 782 patients in the IQR). For the postcycle blastulation analysis, all patients who proceeded to retrieval (whether or not they also had AMH data available) were included (535 patients with oocyte yield in the <10th percentile versus 2675 patients in the IQR). To compare aneuploidy rates, the subset of these patients who pursued PGT-A was then analyzed (156 patients in the <10th percentile versus 1100 patients in the IQR). MAIN RESULTS AND THE ROLE OF CHANCE: The adjusted odds of a given fertilized oocyte developing to a blastocyst, being aneuploid or leading to a live birth after euploid transfer were no different if the oocyte was retrieved from a cycle with ovarian reserve parameters or oocyte yield in the <10th percentile compared to an oocyte retrieved in a cycle with those parameters in the 25-75th percentile. An AMH level in the <10th percentile did more commonly result in cycle cancellation prior to retrieval and after retrieval prior to transfer due to global arrest of embryos. LIMITATIONS, REASONS FOR CAUTION: The timing of retrieval in patients with fewer oocytes may be more optimal given the greater ability to discern the overall maturity of the cohort, thus enhancing performance per retrieved oocyte. Analyses included only first cycles. Subsequent adjustment of protocol due to prior performance may mean that some patients in the <10th percentile for oocyte yield are actually better prognosis patients than their first cycle indicates. Data on whether or not patients were on oral contraceptives at time that AMH level drawn was not available. Other unknown biases are also likely to be present given retrospective nature of the study. WIDER IMPLICATIONS OF THE FINDINGS: While young women with evidence of quantitative depletion of ovarian reserve have lower live birth rates per stimulation cycle, this not attributable to poor oocyte quality because the blastulation rate per fertilized oocyte and live birth rate per embryo transfer are equivalent to that in women with normal quantitative markers of ovarian reserve. Thus, the pathophysiology mediating a premature quantitative decline in ovarian reserve appears different than that which mediates markers of oocyte quality, such as aneuploidy. Young poor responders may use this information to help guide embryo accumulation strategies when considering their family building plans. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Endometrial microbiome at the time of embryo transfer: next-generation sequencing of the 16S ribosomal subunit.

PURPOSE: Characterization of the human microbiome has become more precise with the application of powerful molecular tools utilizing the unique 16S ribosomal subunit's hypervariable regions to greatly increase sensitivity. The microbiome of the lower genital tract can prognosticate obstetrical outcome while the upper reproductive tract remains poorly characterized. Here, the endometrial microbiome at the time of single embryo transfer (SET) is characterized by reproductive outcome. METHODS: Consecutive patients undergoing euploid, SET was included in the analysis. After embryo transfer, performed as per routine, the most distal 5-mm portion of the transfer catheter was sterilely placed in a DNA free PCR tube. Next-generation sequencing of the bacteria specific 16S ribosome gene was performed, allowing genus and species calls for microorganisms. RESULTS: Taxonomy assignments were made on 35 samples from 33 patients and 2 Escherichia coli controls. Of the 33 patients, 18 had ongoing pregnancies and 15 did not. There were a total of 278 different genus calls present across patient samples. The microbiome at time of transfer for those patients with ongoing pregnancy vs. those without ongoing pregnancy was characterized by top genera by sum fraction. Lactobacillus was the top species call for both outcomes. CONCLUSIONS: The data presented here show the microbiome at the time of embryo transfer can successfully be characterized without altering standard clinical practice. This novel approach, both in specimen collection and analysis, is the first step toward the goal of determining physiologic from pathophysiologic microbiota. Further studies will help delineate if differences in the microbiome at the time of embryo transfer have a reliable impact on pregnancy outcome.

Polar body based aneuploidy screening is poorly predictive of embryo ploidy and reproductive potential.

PURPOSE: Polar body (polar body) biopsy represents one possible solution to performing comprehensive chromosome screening (CCS). This study adds to what is known about the predictive value of polar body based testing for the genetic status of the resulting embryo, but more importantly, provides the first evaluation of the predictive value for actual clinical outcomes after embryo transfer. METHODS: SNP array was performed on first polar body, second polar body, and either a blastomere or trophectoderm biopsy, or the entire arrested embryo. Concordance of the polar body-based prediction with the observed diagnoses in the embryos was assessed. In addition, the predictive value of the polar body -based diagnosis for the specific clinical outcome of transferred embryos was evaluated through the use of DNA fingerprinting to track individual embryos. RESULTS: There were 459 embryos analyzed from 96 patients with a mean maternal age of 35.3. The polar body-based predictive value for the embryo based diagnosis was 70.3%. The blastocyst implantation predictive value of a euploid trophectoderm was higher than from euploid polar bodies (51% versus 40%). The cleavage stage embryo implantation predictive value of a euploid blastomere was also higher than from euploid polar bodies (31% versus 22%). CONCLUSION: Polar body based aneuploidy screening results were less predictive of actual clinical outcomes than direct embryo assessment and may not be adequate to improve sustained implantation rates. In nearly one-third of cases the polar body based analysis failed to predict the ploidy of the embryo. This imprecision may hinder efforts for polar body based CCS to improve IVF clinical outcomes.

Embryos whose polar bodies contain isolated reciprocal chromosome aneuploidy are almost always euploid.

STUDY QUESTION: When a chromosome aneuploidy is detected in the first polar body and a reciprocal loss or gain of the same chromosome is detected in the second polar body, is the resulting embryo usually aneuploid for that chromosome? SUMMARY ANSWER: When reciprocal aneuploidy occurs in polar bodies, the resulting embryo is usually normal for that chromosome, indicating that premature separation of sister chromatids (PSSC)-not non-disjunction-likely occurred in meiosis I. WHAT IS KNOWN ALREADY: Single-nucleotide polymorphism-based microarray analysis can be used to accurately determine the chromosomal status of polar bodies and embryos. Sometimes, the only abnormality found is a reciprocal gain or loss of one or two chromosomes in the two polar bodies. Prediction of the status of the resulting embryo in these cases is problematic. STUDY DESIGN, SIZE, DURATION: Blinded microarray analysis of previously diagnosed aneuploid embryos that had reciprocal polar body aneuploidy. MATERIALS, SETTING, METHODS: IVF cycles were performed between 2008 and 2011 in patients aged 40 ± 3 years (range 35-47 years) with an indication for polar body-based aneuploidy screening. Thirty-five aneuploid vitrified Day 3 embryos were warmed, cultured to Day 5 and biopsied for microarray analysis. Predictions were made for the ploidy status of the embryo if PSSC or non-disjunction had occurred. The signal intensity for the aneuploid chromosome in the first polar body was compared between those that resulted in euploid and aneuploid embryos. MAIN RESULTS AND THE ROLE OF CHANCE: Among 34 embryos with evaluable results, 31 were euploid on re-analysis. Of 43 chromosomes that had reciprocal aneuploidy in the polar bodies, 41 were disomic in the embryo, indicating that PSSC was likely to have occurred 95% (95% confidence interval 85-99%) of the time. The log 2 ratio signal intensity from the chromosomes that underwent non-disjunction, resulting in unbalanced embryos, were outliers when compared with those that underwent PSSC. LIMITATIONS, REASONS FOR CAUTION: Although most embryos with reciprocal aneuploid polar bodies were euploid, it is unknown whether they maintain equivalent reproductive potential when transferred. Further study is needed to determine whether these embryos should be re-biopsied and considered for transfer. WIDER IMPLICATIONS OF THE FINDINGS: This study is consistent with increasing evidence that PSSC is the primary cause of meiosis I errors in embryos from women of advanced reproductive age. Clinicians should be cautious in interpreting results from polar body aneuploidy screening, especially when only the first polar body is tested.

Single embryo transfer with comprehensive chromosome screening results in improved ongoing pregnancy rates and decreased miscarriage rates.

BACKGROUND: Single embryo transfer (SET) provides the most certain means to reduce the risk of multiple gestation. Regrettably, prospective trials of SET have demonstrated reductions in per-cycle delivery rates. A validated method of comprehensive chromosome screening (CCS) has the potential to optimize SET by transferring only euploid embryos. This retrospective study evaluates the efficacy of SET with CCS in an infertile population. METHODS: Overall and age-controlled ongoing pregnancy rates (OPR) were compared between women undergoing SET following CCS (CCS-SET, n= 140) and those undergoing SET without aneuploidy screening (control SET, n= 182). All transfers were at the blastocyst stage, with CCS performed after trophectoderm biopsy of expanded blastocysts and analysis with rapid PCR allowing for fresh transfer. RESULTS: In the CCS-SET and control SET groups, an OPR of 55.0 and 41.8%, respectively, was obtained. The OPR was lower for the control group (P< 0.01) despite a younger age than the CCS group (37.3 ± 3.4 versus 34.2 ± 3.9 years; P< 0.001). Birthweight and gestational age at delivery were equivalent. The proportion of clinical pregnancies resulting in miscarriage was higher in the control group (24.8 versus 10.5%, P< 0.01), with more patients requiring surgical interventions for aneuploid pregnancies. There was one monozygotic twin delivery in the CCS group and none in the control group. CONCLUSIONS: Compared with traditional blastocyst SET, SET after trophectoderm biopsy and rapid PCR-based CCS increases OPR and reduces the miscarriage rate. The enhanced selection empowered by CCS with SET may provide a practical way to eliminate multi-zygotic multiple gestation without compromising clinical outcomes per cycle.

SNP microarray-based 24 chromosome aneuploidy screening demonstrates that cleavage-stage FISH poorly predicts aneuploidy in embryos that develop to morphologically normal blastocysts.

Although selection of chromosomally normal embryos has the potential to improve outcomes for patients undergoing IVF, the clinical impact of aneuploidy screening by fluorescence in situ hybridization (FISH) has been controversial. There are many putative explanations including sampling error due to mosaicism, negative impact of biopsy, a lack of comprehensive chromosome screening, the possibility of embryo self-correction and poor predictive value of the technology itself. Direct analysis of the negative predictive value of FISH-based aneuploidy screening for an embryo's reproductive potential has not been performed. Although previous studies have found that cleavage-stage FISH is poorly predictive of aneuploidy in morphologically normal blastocysts, putative explanations have not been investigated. The present study used a single nucleotide polymorphism (SNP) microarray-based 24 chromosome aneuploidy screening technology to re-evaluate morphologically normal blastocysts that were diagnosed as aneuploid by FISH at the cleavage stage. Mosaicism and preferential segregation of aneuploidy to the trophectoderm (TE) were evaluated by characterization of multiple sections of the blastocyst. SNP microarray technology also provided the first opportunity to evaluate self-correction mechanisms involving extrusion or duplication of aneuploid chromosomes resulting in uniparental disomy (UPD). Of all blastocysts evaluated (n = 50), 58% were euploid in all sections despite an aneuploid FISH result. Aneuploid blastocysts displayed no evidence of preferential segregation of abnormalities to the TE. In addition, extrusion or duplication of aneuploid chromosomes resulting in UPD did not occur. These findings support the conclusion that cleavage-stage FISH technology is poorly predictive of aneuploidy in morphologically normal blastocysts.

Free vitamin D does not vary through the follicular phase of the menstrual cycle.

The importance of vitamin D (25OHD) in general health and reproductive success has been a focus in the setting of the 25OHD deficiency epidemic. However, there are challenges to understanding 25OHD's effects. The free and bioavailable levels are affected by 25OHD binding protein (DBP) and it is not known how estradiol fluctuations during the menstrual cycle affect these binding parameters. This may impact the most appropriate time to measure 25OHD when determining deficiency. This study characterizes 25OHD throughout the follicular phase of the menstrual cycle. Patients undergoing natural cycle IVF were included. Serum was drawn throughout the follicular phase of the menstrual cycle; 25OHD, DBP, albumin, and estrogen levels were determined for each time point allowing for mathematical calculation of free and bioavailable 25OHD. Early, mid, and late follicular phases were designated by estrogen tertiles among patients. Mean Levels of 25OHD (total, free, bioavailable) and DBP for each tertile were compared with Kruskil-Wallis test for non-parametric groups. Linear regression with GEE was employed due to repeated measures within participants. A total of 33 patients were included with 202 total serum measurements. There was no difference in mean levels of 25OHD (p = 0.77), free 25OHD (p = 0.91), and bioavailable 25OHD (p = 0.76) when measured throughout the follicular phase of the menstrual cycle. Vitamin D metabolism does not fluctuate as estradiol changes in the follicular phase of the menstrual cycle. This data indicates that assessment of 25OHD, in particular when assessed for associations with reproductive outcomes, can be measured reliably at any point during the follicular phase of the menstrual cycle.

Transforming growth factor-alpha in human implantation trophoblast: immunohistochemical evidence for autocrine/paracrine function.

Epidermal growth factor (EGF) and its receptor (EGF-R) have been demonstrated in human implantation sites. Transforming growth factor-alpha (TGF-alpha), a protein with extensive sequence homology to EGF and with equal affinity for the EGF-R, was localized immunohistochemically in early intrauterine and ectopic pregnancies. Within the same experiments, TGF-alpha immunostaining was more intense in ectopic than intrauterine pregnancies. In both groups, TGF-alpha immunostaining was moderate to intense in the syncytiotrophoblast (ST), light to moderate in the cytotrophoblast (CT), and moderate to intense in intermediate trophoblast (IT). In ST, TGF-alpha immunostaining localized to the cytoplasm and plasma membranes, including microvilli. No nuclear associated TGF-alpha was noted in ST. In CT, differential TGF-alpha immunostaining was noted between the villous and nonvillous CT. Villous CT demonstrated light to absent cytoplasmic TGF-alpha immunostaining with intense nuclear staining. In contrast, nonvillous CT revealed moderate to intense cytoplasmic staining without demonstrable nuclear staining. These results demonstrate the presence of immunoreactive TGF-alpha in all forms of trophoblast. The known presence of the EGF-R suggests an autocrine/paracrine role for TGF-alpha during human implantation.

Immunohistochemical localization of epidermal growth factor in human endometrium, decidua, and placenta.

Epidermal growth factor (EGF) was localized immunohistochemically in human endometrium throughout the menstrual cycle, in gestational decidua, and in first, second, and third trimester placenta using two polyclonal antihuman EGF antisera. In proliferative phase endometrium, moderate EGF immunostaining was localized to the cytoplasm of stromal cells, with absent to light staining of glandular epithelium. In the secretory phase, EGF immunostaining was intense and localized predominantly to stromal cells, particularly those surrounding spiral arterioles. There was absent to light EGF immunostaining within epithelial cells; however, there was no staining of subnuclear vacuoles. In addition, the luminal surface of exhausted secretory glands demonstrated moderate EGF immunostaining. In gestational decidua, EGF immunostaining was light to moderate in the stromal cells, but was intense in the surface epithelium. Intense EGF immunostaining was noted in the syncytiotrophoblast layer of first trimester placenta, with light to moderate staining of the cytotrophoblast. Immunostaining decreased in both layers of trophoblast as pregnancy progressed. Immunoreactive EGF is found in endometrium and trophoblast and may have a physiological role in endometrial and placental function.

Epidermal growth factor and its receptor in human implantation trophoblast: immunohistochemical evidence for autocrine/paracrine function.

Epidermal growth factor (EGF) and its receptor (EGF-R) were immunohistochemically localized in trophoblast during human implantation from intrauterine and ectopic pregnancies. EGF immunostaining was absent to light in the cytotrophoblast (CT), light to moderate in intermediate trophoblast (IT), and intense in the syncytiotrophoblast (ST). In ST, EGF immunostaining was found mostly in the cytoplasm; however, staining of the plasma membrane was also noted. Immunostaining for the EGF-R was absent to light in the CT and moderate to intense in the IT. Immunostaining for the EGF-R was intense in the ST, with moderate staining in the cytoplasm and intense staining in the plasma membrane. Staining was most intense on the microvilli of the ST. Additionally, EGF-R immunostaining could be demonstrated on nuclear membranes. The increase in the intensity of the immunostaining for both EGF and EGF-R noted in CT, IT, and ST suggests a differentiated expression of this receptor-ligand system in human trophoblast and provides evidence for an autocrine/paracrine role for EGF in trophoblast function. The presence of this receptor-ligand system during early human implantation strongly supports a role for EGF and the EGF-R in embryo-uterine signalling and the implantation process.

Immunohistochemical localization of transforming growth factor-alpha in human endometrium, decidua, and trophoblast.

Transforming growth factor-alpha (TGF-alpha) was localized immunohistochemically in human proliferative and secretory endometrium, decidua, and trophoblast from first, second, and third trimester pregnancies. In proliferative endometrium, TGF-alpha immunostaining was moderate to intense and localized predominantly to stromal cells, whereas glandular staining was absent to light. After ovulation, TGF-alpha staining was light within the stroma, but moderate to intense around spiral arterioles. Moderate to intense staining was also detected in glandular and surface epithelium in secretory endometrium, with no staining noted in subnuclear vacuoles. In hypersecretory endometrium, staining was predominantly epithelial. In decidua, TGF-alpha was detected in intermediate trophoblast and on the surface epithelium. In first trimester trophoblast, TGF-alpha was detected in both cytotrophoblast (CT) and syncytiotrophoblast. Cytoplasmic staining was light in CT and moderate to intense in ST, with particular staining of plasma membranes. Intense TGF-alpha staining of nuclear membranes in CT was noted. TGF-alpha staining was light to absent in second and absent in third trimester trophoblast. This study demonstrates immunoreactive TGF-alpha in tissues known to be responsive to epidermal growth factor, and also demonstrates the presence of immunoreactive TGF-alpha associated with nuclear membranes. Thus, TGF-alpha may play an autocrine/paracrine role in endometrial development and trophoblast function.

Immunohistochemical localization of transforming growth factor-beta in human implantation sites.

Transforming growth factor-beta (TGF beta), a protein known to antagonize many of the functions of the epidermal growth factor-receptor system, was localized immunohistochemically in unruptured ectopic pregnancies (EP) removed by salpingectomy (n = 8), uterine decidua from EP (n = 4), and decidua and trophoblast from electively terminated first trimester pregnancies (ETP; n = 8). Two rabbit polyclonal antisera that recognize both TGF beta 1 and beta 2 were used. Immunostaining for TGF beta was identified in all three forms of trophoblast, cytotrophoblasts, intermediate trophoblasts, and syncytiotrophoblasts, which were differentiated histologically and immunohistochemically. Moderate cytoplasmic immunostaining was found in villous cytotrophoblasts in both EP and ETP. Nonvillous (anchoring) cytotrophoblasts in these same tissues demonstrated moderate immunostaining adjacent to the villous and light immunostaining distal to the villous. In intermediate trophoblasts, moderate to intense immunostaining was seen in EP and ETP. Syncytiotrophoblasts demonstrated moderate cytoplasmic immunostaining in EP and ETP as well as moderate to intense staining of plasma membranes and microvilli. Nuclear staining was not evident in any form of trophoblast. TGF beta immunostaining was demonstrated in both glands and stroma of decidua from both EP and ETP; however, staining was more intense in decidua from ETP. With the known presence of TGF beta receptors and mRNA in placenta, these results suggest an autocrine/paracrine role for TGF beta regulation of endometrial-trophoblast function during human implantation.

Hormonal manipulation of endometrial maturation.

Three experimental protocols were devised to induce endometrial maturation in 12 women with ovarian failure. Each was planned to serve a dual purpose: to resolve a particular clinical situation related to synchronization between ovum donor and recipient and to answer a specific question about endometrial physiology. A fourth protocol of sequential estrace (2-6 mg/day) and progesterone (P4; 25-50 mg/day, im) simulating the 28-day natural cycle, served as a control protocol (18 cycles). A short follicular phase protocol consisted of only 6 days of estrogen (E) administration before addition of P4 (13 cycles). In the long follicular phase protocol (5 cycles), estrace was given for 3-5 weeks, and P4 administration was accordingly postponed. In 6 accelerated secretory transformation cycles, 150 mg/day P4 were administered, im, from day 15 onward. The adequacy of the induced endometrial cycles was evaluated by hormonal, morphological, and histochemical criteria relevant to endometrial normalcy and receptivity. Serum estradiol levels and the areas under the estradiol curves for the long and short follicular phase protocols differed significantly from those during the control cycles (P less than 0.005). Areas under the estradiol curves in the accelerated secretory transformation protocol yielded significantly higher P4 values than those in all other protocols (P less than 0.05). All biopsies in the 3 experimental protocols compared favorably with those of the control protocol. Glycocalyx intensity (periodic acid-Schiff) and the amount of galactose residues in the glycocalyx (Ricinus communis-I agglutinin) were greatest during the periimplantation interval. We conclude that a very short exposure of the human endometrium to E or, conversely, prolonged E stimulation will allow normal endometrial maturation with the addition of P4. Supraphysiological doses of P4 in the accelerated secretory transformation protocol significantly enhanced endometrial maturational processes.

Maternal arterial desaturation with 15-methyl prostaglandin F2 alpha for uterine atony.

Over a six-month period, five women with severe uterine atony and postpartum hemorrhage developed marked maternal arterial oxygen desaturation within five to ten minutes of the administration of 15-methyl prostaglandin F2 alpha. The average fall from baseline was 10.4 +/- 5.4%, to a mean arterial oxygen saturation of 88.8 +/- 5.45%. The desaturation was accompanied by acute increases, averaging 20.7 +/- 5.9%, in the intrapulmonary shunt.