Thin endometrial lining: is it more prevalent in patients utilizing preimplantation genetic testing for monogenic disease (PGT-M) and related to prior hormonal contraceptive use?

STUDY QUESTION: Is a thin endometrial lining before ovulation triggering more prevalent in patients utilizing preimplantation genetic testing for monogenic disease (PGT-M) compared to the regular IVF/ICSI population and is this associated with prior hormonal contraceptive use? SUMMARY ANSWER: Thin (<8 mm) endometrial lining is more prevalent in PGT-M patients compared to the regular IVF/ICSI population and is associated with both longer prior hormonal contraceptive use and a shorter cessation interval of hormonal contraceptives before IVF/ICSI treatment. WHAT IS KNOWN ALREADY: Thin endometrial lining has been associated with lower pregnancy rates in IVF/ICSI cycles and increased chances of miscarriage and low birth weight. Endometrial thinning and atrophy occur during hormonal contraceptive use. Patients utilizing PGT-M typically use hormonal contraceptives up until treatment to avoid the risk of conception of a genetically affected child. Whether this could negatively affect endometrial thickness achieved during subsequent IVF/ICSI cycles is not known. STUDY DESIGN, SIZE, DURATION: A retrospective case control study was performed, including all PGT-M patients attending the University Medical Centre Groningen (cases), between 2009 and 2018. The control group consisted of two non-PGT IVF/ICSI patients for each PGT-M patient, matched for age and treatment period. PARTICIPANTS/MATERIALS, SETTING, METHODS: First cycles of 122 PGT-M patients and 240 controls were included. Cessation interval of hormonal contraceptives was categorized as late cessation (cessation <1 year prior to treatment) or early cessation (>1 year prior to treatment). Endometrial thickness was routinely measured on the day of hCG triggering or 1 day prior. The prevalence of an endometrial lining <8 mm was compared between PGT-M patients and controls. Hormonal contraceptive use (both duration and cessation interval) was compared between both groups. Univariable and multivariable regression analyses were performed to identify risk factors for thin endometrial lining. In addition, cycle and pregnancy outcomes were compared within control/PGT-M groups between patients with endometrial lining > or <8 mm. MAIN RESULTS AND THE ROLE OF CHANCE: Thin endometrial lining on the day of hCG triggering was found significantly more often in the PGT-M group, compared to controls: 32% vs 11% (mean difference 21.0%, 95% CI: 11.7, 30.3%). As expected, more patients in the PGT-M group ceased their hormonal contraception late (<1 year): 64% vs 2% in the control group (mean difference 61.9%, 95% CI: 53.0, 70.8%). Average duration of hormonal contraceptive use was 10.6 years in the PGT-M group vs 9.3 years in controls (mean difference 1.3 years, 95% CI: 0.2, 2.3 years). Multivariable logistic regression analysis identified late cessation (OR: 6.0, 95% CI: 1.9-19.2) and duration of prior hormonal contraceptive use (OR per year increase 1.1, 95% CI: 1.0-1.2) as significant independent risk factors for a thin endometrial lining. In relation to outcome, we found a statistically significant increase in miscarriage rate in PGT-M patients with an endometrial lining <8 mm compared to those with an endometrial lining >8 mm (20.0% vs 1.7%, mean difference 18.3%, 95% CI: 2.3, 34.3%). A trend towards lower birth weight and gestation- and gender-adjusted birth weight (z-score) was also found in this group. No statistically significant differences were detected in pregnancy rate, live birth rate, or incidence of preterm delivery or SGA. Within the control group, no statistically significant differences were found in outcomes between patients with an endometrial lining <8 compared to an endometrial lining >8 mm. LIMITATIONS, REASONS FOR CAUTION: The study is retrospective. Various types of hormonal contraceptives were reported which possibly exert different effects on the endometrial lining. In relation to pregnancy outcome measures, numbers were very limited; therefore, no firm conclusions should be drawn. WIDER IMPLICATIONS OF THE FINDINGS: This study provides further insight into the role of prior hormonal contraceptive use as a possible contributor to the occurrence of thin endometrial lining during ART treatment. Future studies should provide more information on its clinical relevance, to determine whether PGT-M patients can be reassured, or should be counselled to stop hormonal contraceptive use and change to an alternative contraceptive method prior to PGT treatment. STUDY FUNDING/COMPETING INTERESTS: No specific funding was used and no conflicts of interests are declared. TRIAL REGISTRATION NUMBER: N/A.

Trimethylamine-N-oxide is present in human follicular fluid and is a negative predictor of embryo quality.

STUDY QUESTION: Are levels of trimethylamine-N-oxide (TMAO) in human follicular fluid (FF) related to IVF outcomes? SUMMARY ANSWER: Higher levels of TMAO are a negative predictor of oocyte fertilization and embryo quality. WHAT IS KNOWN ALREADY: TMAO is a metabolic product of dietary choline and l-carnitine produced via subsequent enzymatic modifications by the intestinal microbiota and hepatocytes. TMAO promotes inflammatory and oxidative stress pathways and has been characterized as a causative biomarker for the development of cardiometabolic disease. STUDY DESIGN, SIZE, DURATION: For the present cross-sectional study, samples (FF and plasma) from 431 modified natural cycle (MNC)-IVF cycles of 132 patients were collected prospectively between October 2014 and March 2018 in a single academic medical center. PARTICIPANTS/MATERIALS, SETTING, METHODS: TMAO and its precursors (choline, l-carnitine and gamma-butyrobetaine) were measured by ultra-high-performance liquid chromatography/mass spectrometry in (i) matched FF and plasma from 63 MNC-IVF cycles, in order to compare metabolite levels in the two matrices and (ii) FF from 232 MNC-IVF cycles in which only one oocyte was retrieved at follicular puncture. The association between metabolite levels and oocyte fertilization, embryo fragmentation percentage, embryo quality and the occurrence of pregnancy was analyzed using multilevel generalized estimating equations with adjustment for patient and cycle characteristics. MAIN RESULTS AND THE ROLE OF CHANCE: The level of choline was higher in FF as compared to matched plasma (P < 0.001). Conversely, the levels of TMAO and gamma-butyrobetaine were lower in FF as compared to plasma (P = 0.001 and P = 0.075, respectively). For all metabolites, there was a positive correlation between FF and plasma levels. Finally, levels of TMAO and its gut-derived precursor gamma-butyrobetaine were lower in FF from oocytes that underwent normal fertilization (TMAO: odds ratio [OR] 0.66 [0.49-0.90], P = 0.008 per 1.0-µmol/L increase; gamma-butyrobetaine: OR 0.77 [0.60-1.00], P = 0.047 per 0.1-µmol/L increase) and developed into top-quality embryos (TMAO: OR 0.56 [0.42-0.76], P < 0.001 per 1.0-µmol/L increase; gamma-butyrobetaine: OR 0.79 [0.62-1.00], P = 0.050 per 0.1-µmol/L increase) than in FF from oocytes of suboptimal development. LIMITATIONS, REASONS FOR CAUTION: The individual contributions of diet, gut bacteria and liver to the metabolite pools have not been quantified in this analysis. WIDER IMPLICATIONS OF THE FINDINGS: More research on the contribution of diet and the effect of gut bacteria on FF TMAO is warranted. Since TMAO integrates diet, microbiota and genetic setup of the person, our results indicate potential important clinical implications for its use as biomarker for lifestyle interventions to improve fertility. STUDY FUNDING/COMPETING INTEREST(S): No external funding was received for this project. The Department of Obstetrics and Gynecology of the University Medical Center Groningen received an unrestricted educational grant of Ferring Pharmaceutical BV, the Netherlands. The authors have no other conflicts of interest. TRIAL REGISTRATION NUMBER: Netherlands Trial Register number NTR4409.

BRCA1 mutation carriers have a lower number of mature oocytes after ovarian stimulation for IVF/PGD.

PURPOSE: The aim of this study was to determine whether BRCA1/2 mutation carriers produce fewer mature oocytes after ovarian stimulation for in vitro fertilization (IVF) with preimplantation genetic diagnosis (PGD), in comparison to a PGD control group. METHODS: A retrospective, international, multicenter cohort study was performed on data of first PGD cycles performed between January 2006 and September 2015. Data were extracted from medical files. The study was performed in one PGD center and three affiliated IVF centers in the Netherlands and one PGD center in Belgium. Exposed couples underwent PGD because of a pathogenic BRCA1/2 mutation, controls for other monogenic conditions. Only couples treated in a long gonadotropin-releasing hormone (GnRH) agonist-suppressive protocol, stimulated with at least 150 IU follicle stimulating hormone (FSH), were included. Women suspected to have a diminished ovarian reserve status due to chemotherapy, auto-immune disorders, or genetic conditions (other than BRCA1/2 mutations) were excluded. A total of 106 BRCA1/2 mutation carriers underwent PGD in this period, of which 43 (20 BRCA1 and 23 BRCA2 mutation carriers) met the inclusion criteria. They were compared to 174 controls selected by frequency matching. RESULTS: Thirty-eight BRCA1/2 mutation carriers (18 BRCA1 and 20 BRCA2 mutation carriers) and 154 controls proceeded to oocyte pickup. The median number of mature oocytes was 7.0 (interquartile range (IQR) 4.0-9.0) in the BRCA group as a whole, 6.5 (IQR 4.0-8.0) in BRCA1 mutation carriers, 7.5 (IQR 5.5-9.0) in BRCA2 mutation carriers, and 8.0 (IQR 6.0-11.0) in controls. Multiple linear regression analysis with the number of mature oocytes as a dependent variable and adjustment for treatment center, female age, female body mass index (BMI), type of gonadotropin used, and the total dose of gonadotropins administered revealed a significantly lower yield of mature oocytes in the BRCA group as compared to controls (p = 0.04). This finding could be fully accounted for by the BRCA1 subgroup (BRCA1 mutation carriers versus controls p = 0.02, BRCA2 mutation carriers versus controls p = 0.50). CONCLUSIONS: Ovarian response to stimulation, expressed as the number of mature oocytes, was reduced in BRCA1 but not in BRCA2 mutation carriers. Although oocyte yield was in correspondence to a normal response in all subgroups, this finding points to a possible negative influence of the BRCA1 gene on ovarian reserve.

Presence of bile acids in human follicular fluid and their relation with embryo development in modified natural cycle IVF.

STUDY QUESTION: Are bile acids (BA) and their respective subspecies present in human follicular fluid (FF) and do they relate to embryo quality in modified natural cycle IVF (MNC-IVF)? SUMMARY ANSWER: BA concentrations are 2-fold higher in follicular fluid than in serum and ursodeoxycholic acid (UDCA) derivatives were associated with development of top quality embryos on Day 3 after fertilization. WHAT IS KNOWN ALREADY: Granulosa cells are capable of synthesizing BA, but a potential correlation with oocyte and embryo quality as well as information on the presence and role of BA subspecies in follicular fluid have yet to be investigated. STUDY DESIGN, SIZE, DURATION: Between January 2001 and June 2004, follicular fluid and serum samples were collected from 303 patients treated in a single academic centre that was involved in a multicentre cohort study on the effectiveness of MNC-IVF. PARTICIPANTS/MATERIALS, SETTING, METHODS: Material from patients who underwent a first cycle of MNC-IVF was used. Serum was not stored from all patients, and the available material comprised 156 follicular fluid and 116 matching serum samples. Total BA and BA subspecies were measured in follicular fluid and in matching serum by enzymatic fluorimetric assay and liquid chromatography-mass spectrometry, respectively. The association of BA in follicular fluid with oocyte and embryo quality parameters, such as fertilization rate and cell number, presence of multinucleated blastomeres and percentage of fragmentation on Day 3, was analysed. MAIN RESULTS AND THE ROLE OF CHANCE: Embryos with eight cells on Day 3 after oocyte retrieval were more likely to originate from follicles with a higher level of UDCA derivatives than those with fewer than eight cells (P < 0.05). Furthermore, follicular fluid levels of chenodeoxycholic derivatives were higher and deoxycholic derivatives were lower in the group of embryos with fragmentation compared with those without (each P < 0.05). Levels of total BA were 2-fold higher in follicular fluid compared with serum (P < 0.001), but had no predictive value for oocyte and embryo quality. LIMITATIONS, REASONS FOR CAUTION: Only samples originating from first cycle MNC-IVF were used, which resulted in 14 samples only from women with an ongoing pregnancy, therefore further prospective studies are required to confirm the association of UDCA with IVF pregnancy outcomes. The inter-cycle variability of BA levels in follicular fluid within individuals has yet to be investigated. We checked for macroscopic signs of contamination of follicular fluid by blood but the possibility that small traces of blood were present within the follicular fluid remains. Finally, although BA are considered stable when stored at -20°C, there was a time lag of 10 years between the collection and analysis of follicular fluid and serum samples. WIDER IMPLICATIONS OF THE FINDINGS: The favourable relation between UDCA derivatives in follicular fluid and good embryo development and quality deserves further prospective research, with live birth rates as the end-point. STUDY FUNDING/COMPETING INTERESTS: This work was supported by a grant from the Netherlands Organisation for Scientific Research (VIDI Grant 917-56-358 to U.J.F.T.). No competing interests are reported.

NKL homeobox genes in leukemia.

NK-like (NKL) homeobox genes code for transcription factors, which can act as key regulators in fundamental cellular processes. NKL genes have been implicated in divergent types of cancer. In this review, we summarize the involvement of NKL genes in cancer and leukemia in particular. NKL genes can act as tumor-suppressor genes and as oncogenes, depending on tissue type. Aberrant expression of NKL genes is especially common in T-cell acute lymphoblastic leukemia (T-ALL). In T-ALL, 8 NKL genes have been reported to be highly expressed in specific T-ALL subgroups, and in ~30% of cases, high expression is caused by chromosomal rearrangement of 1 of 5 NKL genes. Most of these NKL genes are normally not expressed in T-cell development. We hypothesize that the NKL genes might share a similar downstream effect that promotes leukemogenesis, possibly due to mimicking a NKL gene that has a physiological role in early hematopoietic development, such as HHEX. All eight NKL genes posses a conserved Eh1 repressor motif, which has an important role in regulating downstream targets in hematopoiesis and possibly in leukemogenesis as well. Identification of a potential common leukemogenic NKL downstream pathway will provide a promising subject for future studies.

NOTCH1 and/or FBXW7 mutations predict for initial good prednisone response but not for improved outcome in pediatric T-cell acute lymphoblastic leukemia patients treated on DCOG or COALL protocols.

Aberrant activation of the NOTCH1 pathway by inactivating and activating mutations in NOTCH1 or FBXW7 is a frequent phenomenon in T-cell acute lymphoblastic leukemia (T-ALL). We retrospectively investigated the relevance of NOTCH1/FBXW7 mutations for pediatric T-ALL patients enrolled on Dutch Childhood Oncology Group (DCOG) ALL7/8 or ALL9 or the German Co-Operative Study Group for Childhood Acute Lymphoblastic Leukemia study (COALL-97) protocols. NOTCH1-activating mutations were identified in 63% of patients. NOTCH1 mutations affected the heterodimerization, the juxtamembrane and/or the PEST domains, but not the RBP-J-κ-associated module, the ankyrin repeats or the transactivation domain. Reverse-phase protein microarray data confirmed that NOTCH1 and FBXW7 mutations resulted in increased intracellular NOTCH1 levels in primary T-ALL biopsies. Based on microarray expression analysis, NOTCH1/FBXW7 mutations were associated with activation of NOTCH1 direct target genes including HES1, DTX1, NOTCH3, PTCRA but not cMYC. NOTCH1/FBXW7 mutations were associated with TLX3 rearrangements, but were less frequently identified in TAL1- or LMO2-rearranged cases. NOTCH1-activating mutations were less frequently associated with mature T-cell developmental stage. Mutations were associated with a good initial in vivo prednisone response, but were not associated with a superior outcome in the DCOG and COALL cohorts. Comparing our data with other studies, we conclude that the prognostic significance for NOTCH1/FBXW7 mutations is not consistent and may depend on the treatment protocol given.

Cooperative genetic defects in TLX3 rearranged pediatric T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder, in which multiple genetic abnormalities cooperate in the malignant transformation of thymocytes. About 20% of pediatric T-ALL cases are characterized by TLX3 expression due to a cryptic translocation t(5;14)(q35;q32). Although a number of collaborating genetic events have been identified in TLX3 rearranged T-ALL patients (NOTCH1 mutations, p15/p16 deletions, NUP214-ABL1 amplifications), further elucidation of additional genetic lesions could provide a better understanding of the pathogenesis of this specific T-ALL subtype. In this study, we used array-CGH to screen TLX3 rearranged T-ALL patients for new chromosomal imbalances. Array-CGH analysis revealed five recurrent genomic deletions in TLX3 rearranged T-ALL, including del(1)(p36.31), del(5)(q35), del(13)(q14.3), del(16)(q22.1) and del(19)(p13.2). From these, the cryptic deletion, del(5)(q35), was exclusively identified in about 25% of TLX3 rearranged T-ALL cases. In addition, 19 other genetic lesions were detected once in TLX3 rearranged T-ALL cases, including a cryptic WT1 deletion and a deletion covering the FBXW7 gene, an U3-ubiquitin ligase that mediates the degradation of NOTCH1, MYC, JUN and CyclinE. This study provides a genome-wide overview of copy number changes in TLX3 rearranged T-ALL and offers great new challenges for the identification of new target genes that may play a role in the pathogenesis of T-ALL.

Sustained and transient attention in the continuous performance task.

One of the most frequently applied methods to study abnormal cognition is the Continuous Performance Task (CPT). It is unclear, however, which cognitive functions are engaged in normal CPT performance. The aims of the present study were to identify the neurocognitive functions engaged in the main variants of the CPT and to determine to what extent these variants differentially engage these functions. We hypothesized that the main CPT versions (CPT-X, CPT-AX, CPT-Identical Pairs) can be distinguished by whether they demand sustained or transient attention and sustained or transient response preparation. Transient attention to objects like letters or digits, that is, the need to switch attention to different objects from trial to trial, impairs target detection accuracy relative to sustained attention to a single object. Transient response preparation, that is, the possibility to switch response preparation on and off from trial to trial, improves response speed relative to having to sustain response preparation across all trials. Comparison of task performance and Event-Related brain Potentials (ERPs) of healthy participants obtained in the main CPT variants confirmed these hypotheses. Behavioral and ERP measures indicated worse target detection in the CPT-AX than in the CPT-X, consistent with a higher demand on transient attention in that task. In contrast, behavioral and ERP measures indicated higher response speed in the CPT-AX than in the CPT-X, associated with more response preparation in advance of the targets. This supports the idea of increased transient response preparation in the CPT-AX. We conclude that CPTs differ along at least two task variables that each influences a different cognitive function.