Evaluation of Nanopore Sequencing on Polar Bodies for Routine Pre-Implantation Genetic Testing for Aneuploidy.

BACKGROUND: Preimplantation genetic testing for aneuploidy (PGT-A) using polar body (PB) biopsy offers a clinical benefit by reducing the number of embryo transfers and miscarriage rates but is currently not cost-efficient. Nanopore sequencing technology opens possibilities by providing cost-efficient and fast sequencing results with uncomplicated sample preparation work flows. METHODS: In this comparative experimental study, 102 pooled PB samples (99 passing QC) from 20 patients were analyzed for aneuploidy using nanopore sequencing technology and compared with array comparative genomic hybridization (aCGH) results generated as part of the clinical routine. Samples were sequenced on a Nanopore MinION machine. Whole-chromosome copy-numbers were called by custom bioinformatic analysis software. Automatically called results were compared to aCGH results. RESULTS: Overall, 96/99 samples were consistently detected as euploid or aneuploid in both methods (concordance = 97.0%, sensitivity = 0.957, specificity = 1.0, positive predictive value = 1.0, negative predictive value = 0.906). On the chromosomal level, concordance reached 98.7%. Chromosomal aneuploidies analyzed in this trial covered all 23 chromosomes with 98 trisomies, and 97 monosomies in 70 aCGH samples.The whole nanopore work flow is feasible in under 5 h (for one sample) with a maximum time of 16 h (for 12 samples), enabling fresh PB-euploid embryo transfer. A material cost of US$ 165 (EUR 150)/sample possibly enables cost-efficient aneuploidy screening. CONCLUSIONS: This is the first study systematically comparing nanopore sequencing with standard methods for the detection of PB aneuploidy. High concordance rates confirmed the feasibility of nanopore technology for this application. Additionally, the fast and cost-efficient work flow reveals the clinical utility of this technology, making it clinically attractive for PB PGT-A.

Comment: pregnancy after bariatric surgery - achievements and open questions.

Bariatric surgery confers potential advantages for obese patients, but also risks for pregnancy. Perinatal outcomes may be varying between surgical procedures. This topic was recently addressed by a systematic review in BMC Pregnancy and Childbirth. This commentary will discuss the scientific background and implications for future research.

16S rRNA long-read nanopore sequencing is feasible and reliable for endometrial microbiome analysis.

RESEARCH QUESTION: Full-length 16S rRNA gene sequencing using nanopore technology is a fast alternative to conventional short-read 16S rRNA gene sequencing with low initial investment costs that has been used for various microbiome studies but has not yet been investigated as an alternative approach for endometrial microbiome analysis. Is in-situ 16S rRNA gene long-read sequencing using portable nanopore sequencing technology feasible and reliable for endometrial microbiome analysis? DESIGN: A prospective experimental study based on 33 patients seeking infertility treatment between January and October 2019. A 16S rRNA gene long-read nanopore sequencing protocol for analysing endometrial microbiome samples was established, including negative controls for contamination evaluation and positive controls for bias evaluation. Contamination caused by kit and exterior sources was identified and excluded from the analysis. Endometrial samples from 33 infertile patients were sequenced using the optimized long-read nanopore sequencing protocol and compared with conventional short-read sequencing carried out by external laboratories. RESULTS: Of the 33 endometrial patient samples, 23 successfully amplified (69.7%) and their microbiome was assessed using nanopore sequencing. Of those 23 samples, 14 (60.9%) were Lactobacillus-dominated (>80% of reads mapping to Lactobacillus), with 10 samples resulting in more than 90% Lactobacillus reads. Our long-read nanopore sequencing revealed results similar to two conventional short-read sequencing approaches and to long-read sequencing validation carried out in external laboratories. CONCLUSION: In this pilot study, 16S rRNA gene long-read nanopore sequencing was established to analyse the endometrial microbiome in situ that could be widely applied owing to its cost efficiency and portable character.

Intrauterine Fetal Growth Delay During Late Pregnancy After Maternal Gastric Bypass Surgery.

PURPOSE: To investigate intrauterine fetal growth development and birth anthropometry of fetuses conceived after maternal gastric bypass surgery. MATERIALS AND METHODS: Longitudinal cohort study describing longitudinal growth estimated by ultrasound on 43 singleton pregnancies after gastric bypass compared to 43 BMI-matched controls. RESULTS: In fetuses after maternal gastric bypass surgery, growth percentiles decreased markedly from the beginning of the second trimester until the end of the third trimester (decrease of 3.1 fetal abdomen circumference percentiles (95 %CI 0.9-5.3, p = 0.007) per four gestational weeks). While in the second trimester, fetal anthropometric measures did not differ between the groups, the mean abdomen circumference percentiles appeared significantly smaller during the third trimester in offspring of mothers after gastric bypass (mean difference 25.1 percentiles, p < 0.001). Similar tendencies have been observed in estimated fetal weight resulting in significantly more SGA offspring at delivery in the gastric bypass group. In children born after maternal gastric bypass surgery, weight percentiles (32.12th vs. 55.86th percentile, p < 0.001) as well as placental weight (525.2 g vs. 635.7 g, p < 0.001) were significantly reduced compared to controls. CONCLUSION: In fetuses conceived after maternal gastric bypass, intrauterine fetal growth distinctively declined in the second and third trimester, most prominently observed in fetal abdomen circumferences. Birth weight and placental weight at birth was significantly lower compared to BMI-matched controls, possibly due to altered maternal metabolic factors and comparable to mothers experiencing chronic hunger episodes.

Heavy black tea consumption and elevated CA 19-9 and CA 125 levels. A case report on a patient with ovarian endometriotic cysts.

In this case report, we present a case of false positive CA 19-9 and CA 125 levels in a patient with suspected endometriotic cysts. The patient is a 34-year-old nulliparous woman with heavy black tea consumption and elevated CA 19-9 and CA 125 levels. After discontinuation of black tea intake and careful exploration of other possible factors, CA 19-9 and CA 125 levels dropped markedly. As a conclusion, heavy black tea consumption can lead to false positive results of elevated CA 19-9 and CA 125 levels.

Endometriosis and cumulative live birth rate after fresh and frozen IVF cycles with single embryo transfer in young women: no impact beyond reduced ovarian sensitivity-a case control study.

PURPOSE: To investigate the impact of symptomatic and surgically confirmed endometriosis on ovarian sensitivity index (OSI) and cumulative live-birth rates (LBR) using predominantly single embryo transfer (SET). METHODS: Cross-sectional case-control study in a University-based ART program. Women with symptomatic and surgically confirmed endometriosis (N = 172), who underwent IVF/ICSI at Karolinska University Hospital were compared to controls without clinically suspected endometriosis (N = 2585). Two thousand seven hundred fifty-seven patients underwent 8236 treatment cycles (4598 fresh and 3638 frozen cycles). Primary outcome measures included Ovarian Sensitivity Index (OSI) estimated as collected oocytes/FSH dose and cumulative LBR/oocyte pickup (OPU). Generalized estimated equation (GEE) model accounting for dependencies between consecutive treatments were applied. Secondary outcomes included number of oocytes, pregnancy rate per OPU and per ET, LBR per ET, and miscarriage rate. RESULTS: Patients diagnosed with endometriosis had significantly fewer oocytes collected (8.47 vs. 9.54, p = 0.015) and lower OSI (p = 0.011) than controls. There were no differences in cycle cancelations (p = 0.59) or miscarriages (p = 0.95) between the two groups. Cumulative LBR/OPU did not differ between women with endometriosis and controls (35.6% vs. 34.7%, respectively, p = 0.83). In both groups, more than 60% of women had consecutive FETs after fresh ETs (p = 0.49) with SET in > 70% of cases. The results were similar whether ovarian endometrioma was present or not. CONCLUSIONS: Our data support that a diagnosis of endometriosis, with or without present endometrioma, does not negatively affect ART cumulative results. The impact of endometriosis was discernible on OSI but not on clinical relevant outcomes including pregnancy and LBR.

Late metabolic precursors for selective aromatic residue labeling.

In recent years, we developed a toolbox of heavy isotope containing compounds, which serve as metabolic amino acid precursors in the E. coli-based overexpression of aromatic residue labeled proteins. Our labeling techniques show excellent results both in terms of selectivity and isotope incorporation levels. They are additionally distinguished by low sample production costs and meet the economic demands to further implement protein NMR spectroscopy as a routinely used method in drug development processes. Different isotopologues allow for the assembly of optimized protein samples, which fulfill the requirements of various NMR experiments to elucidate protein structures, analyze conformational dynamics, or probe interaction surfaces. In the present article, we want to summarize the precursors we developed so far and give examples of their special value in the probing of protein-ligand interaction.

Morphology of early intrauterine deaths with full trisomy 15.

OBJECTIVE: The morphologic features of embryos with full trisomy 15 are described. METHOD: A total of 1195 pregnancy losses were examined embryoscopically and cytogenetically. RESULTS: Of 1173 successfully karyotyped specimens, full trisomy 15 was diagnosed cytogenetically in 59 cases (5%). All 59 trisomy 15 embryos were diagnosed cytogenetically in the group of 962 embryonic miscarriages (6%). Trisomy 15 was not registered in 171 anembryonic or yolk sac miscarriages, and no case of full trisomy 15 was observed in 62 fetal miscarriages. Fifty-eight embryos with full trisomy 15 showed structural defects on embryoscopic examination. The most common defects were craniofacial anomalies (n = 73), retarded development of the limbs (n = 39), and abnormally short umbilical cords closely attaching the embryo to the chorionic plate (n = 27). Seven embryos were classified as growth disorganized. Limb reduction defects with a prevalence of 5.6/10 000 births, all affecting upper limb development (10 terminal transverse limb reduction defects and 3 embryos with split hand), were registered in 13 (22%) trisomy 15 embryos. CONCLUSION: Limb reduction defects and craniofacial abnormalities are a typical feature of trisomy 15. Gene dosage imbalances related to trisomy 15 might be the main molecular mechanism underlying the developmental defects observed in the present study and require further investigation.

Pregnancy after bariatric surgery: a narrative literature review and discussion of impact on pregnancy management and outcome.

Bariatric surgery (BS) is regarded to be the most effective treatment of obesity with long lasting beneficial effects including weight loss and improvement of metabolic disorders. A considerable number of women undergoing BS are at childbearing age.Although the surgery mediated weight loss has a positive effect on pregnancy outcome, the procedures might be associated with adverse outcomes as well, for example micronutrient deficiencies, iron or B12 deficiency anemia, dumping syndrome, surgical complications such as internal hernias, and small for gestational age (SGA) offspring, possibly due to maternal undernutrition. Also, there is no international consensus concerning the ideal time to conception after BS. Hence, the present narrative review intents to summarize the available literature concerning the most common challenges which arise before and during pregnancy after BS, such as fertility related considerations, vitamin and nutritional deficiencies and their adequate compensation through supplementation, altered glucose metabolism and its implications for gestational diabetes screening, the symptoms and treatment of dumping syndrome, surgical complications and the impact of BS on pregnancy outcome. The impact of different bariatric procedures on pregnancy and fetal outcome will also be discussed, as well as general considerations concerning the monitoring and management of pregnancies after BS.Whereas BS leads to the mitigation of many obesity-related pregnancy complications, such as gestational diabetes mellitus (GDM), pregnancy induced hypertension and fetal macrosomia; those procedures pose new risks which might lead to adverse outcomes for mothers and offspring, for example nutritional deficiencies, anemia, altered maternal glucose metabolism and small for gestational age children.

Pre- and peripartal management of a woman with McArdle disease: a case report.

McArdle disease or glycogen storage disease (GSD) type V is a rare autosomal recessive inherited disorder in skeletal muscle metabolism leading to exercise intolerance, muscle cramps and in some cases to rhabdomyolysis and acute renal failure due to elevated serum myoglobin levels. Albeit the uterine smooth muscle is not affected, pregnancy and delivery can be physically strenuous and may require specific anesthesiologic care. However, data on pregnancy progress and outcome and on special implications linked to anesthesia in women with McArdle's disease is scarce, thus posing a challenge to pre- and peripartal management. We report a case of a pregnant woman with Morbus McArdle who was monitored during her pregnancy and delivered a healthy male via cesarean section under spinal anesthesia. Pregnancy, delivery and recovery were uneventful. Our findings, combined with a literature review, lead to the conclusion that uncomplicated pregnancy and delivery can be expected.

Allogeneic ovarian transplantation using immunomodulator preimplantation factor (PIF) as monotherapy restored ovarian function in olive baboon.

PURPOSE: Allogeneic ovarian transplantation may be an alternative in the future to oocyte donation in women with premature ovarian failure. The objectives of this study were to (a) evaluate allotransplantation feasibility for restoration of ovarian function and (b) assess efficacy of synthetic preimplantation factor (PIF) monotherapy as sole immune-acceptance regimen. METHODS: This is an experimental animal study using non-human primates (Papio anubis). Allogeneic orthotopic ovarian tissue transplantation was performed in two female olive baboons. PIF was administered as a monotherapy to prevent immune rejection and achieve transplant maintenance and function. Subjects underwent bilateral oophorectomy followed by cross-transplantation of prepared ovarian cortex. Postoperatively, subjects were monitored for clinical and biochemical signs of graft rejection and return of function. Weekly blood samples were obtained to monitor graft acceptance and endocrine function restoration. RESULTS: Postoperatively, there were no clinical signs of rejection. Laboratory parameters (alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine) did not indicate organ rejection at any stage of the experiment. Initially, significant loss of follicles was noticed after grafting and serum follicle-stimulating hormone (FSH) and E2 levels were consistent with ovarian failure. Seven months after transplantation, one animal exhibited recurrence of ovarian endocrine function (perineal swelling, E2 rise, FSH decrease, and return of menstruation). CONCLUSIONS: Organ rejection after allogeneic ovarian transplantation was prevented using PIF as monotherapy for the first time and no side effects were recorded. The study suggests the clinical feasibility of ovarian allotransplantation to obtain ovarian function.

Altered glucose profiles and risk for hypoglycaemia during oral glucose tolerance testing in pregnancies after gastric bypass surgery.

AIMS/HYPOTHESIS: A history of gastric bypass surgery can influence the results of the OGTT recommended during pregnancy. Therefore, we compared OGTT glucose kinetics and pregnancy outcome between pregnant gastric bypass patients and BMI-matched, lean and obese controls. METHODS: Medical records were used to collect data on glucose measurements during the 2 h 75 g OGTT as well as on pregnancy and fetal outcome for 304 women (n = 76 per group, matched for age and date of delivery). RESULTS: Women after bariatric surgery had lower fasting glucose levels compared with lean, obese and BMI-matched controls, and showed altered postprandial glucose kinetics, including a rise at 60 min followed by hypoglycaemia with serum glucose of <3.34 mmol/l (which occurred in 54.8%). Moreover, their risk of pre-eclampsia or gestational hypertension was reduced, with an increased risk of delivering small for gestational age infants. CONCLUSIONS/INTERPRETATION: Alternative strategies to accurately define impaired glucose metabolism in pregnancies after bariatric surgery should be explored.

Non-invasive preimplantation genetic screening using array comparative genomic hybridization on spent culture media: a proof-of-concept pilot study.

The aim of this pilot study was to assess if array comparative genomic hybridization (aCGH), non-invasive preimplantation genetic screening (PGS) on blastocyst culture media is feasible. Therefore, aCGH analysis was carried out on 22 spent blastocyst culture media samples after polar body PGS because of advanced maternal age. All oocytes were fertilized by intracytoplasmic sperm injection and all embryos underwent assisted hatching. Concordance of polar body analysis and culture media genetic results was assessed. Thirteen out of 18 samples (72.2%) revealed general concordance of ploidy status (euploid or aneuploid). At least one chromosomal aberration was found concordant in 10 out of 15 embryos found to be aneuploid by both polar body and culture media analysis. Overall, 17 out of 35 (48.6%) single chromosomal aneuploidies were concordant between the culture media and polar body analysis. By analysing negative controls (oocytes with fertilization failure), notable maternal contamination was observed. Therefore, non-invasive PGS could serve as a second matrix after polar body or cleavage stage PGS; however, in euploid results, maternal contamination needs to be considered and results interpreted with caution.

Weekend-free scheduled IVF/ICSI procedures and single embryo transfer do not reduce live-birth rates in a general infertile population.

INTRODUCTION: Scheduling of ovum pickup only on weekdays may result in cases of apparently suboptimal timing for human chorionic gonadotropin and ovum pickup. This study aimed to assess whether live-birth rates were reduced in women with a potentially suboptimal day for human chorionic gonadotropin and ovum pickup to avoid weekend work, according to ultrasonographic data on the day of human chorionic gonadotropin planning. MATERIAL AND METHODS: An evaluation of the optimal human chorionic gonadotropin priming date was performed in treatment protocols of 1000 consecutive patients undergoing their first in vitro fertilization/intracytoplasmatic sperm injection with single-embryo transfer. An ideal ovum pickup day was characterized by human chorionic gonadotropin-scheduling when three or more follicles reached 17 mm (day 0) or with one day of delay (day +1) (n = 760). A non-ideal ovum pickup was either early (day -1, -2, -3) (n = 24) or delayed (day +2, +3, +4) (n = 216). Live-birth rates in the ideal and non-ideal ovum pickup groups was set as primary outcome measure. RESULTS: Early-ovum pickups were excluded as they were infrequent. No differences between ideal and delayed ovum pickup groups were found regarding number of oocytes retrieved (9.87 vs. 9.78, p = 0.990), pregnancy rates (28.3% vs. 29.6%, p = 0.701) or live-birth rates (26.2% vs. 25.9%, p = 0.939). However, sub analyses indicated that treatment with gonadotropin releasing hormone antagonists resulted in significantly lower clinical pregnancy rates in delayed ovum pickups (odds ratio 0.46, p = 0.014), compared with agonist treatments. CONCLUSIONS: Weekend work may not be needed for in vitro fertilization/intracytoplasmatic sperm injection single-embryo transfer treatments. However, in gonadotropin releasing hormone antagonist cycles, delaying ovum pickup more than one day may result in unfavorable outcomes.

Reproductive outcome in European and Middle Eastern/North African patients.

The aim of this retrospective cohort study was to assess differences in infertility-related baseline characteristics and IVF outcome between European and Middle Eastern/North African (MENA) patients. Of 2703 patients undergoing their first IVF cycle, 2485 were Caucasian of European descent and 218 originated from the MENA region. MENA patients were significantly younger (30.6 versus 34.0 years, P < 0.001), less likely smokers, with higher body mass indexes. Infertility duration was longer in MENA patients (P < 0.001), their male partners were younger (P < 0.001) and smoked more often than European male patients (P = 0.005). Male factor infertility (P = 0.017) and polycystic ovary syndrome (PCOS; P = 0.032) was more prevalent in MENA patients, showed significantly higher basal FSH concentrations (P = 0.012) and significantly fewer oocytes retrieved (RR 0.83, 95% CI 0.74-0.93, P = 0.001). Clinical pregnancy rates were comparable (22.4% [European] versus 22.9% [MENA]). Fewer MENA patients had surplus embryos cryopreserved (OR 0.41, 95% CI 0.22-0.76, P = 0.004). Despite younger age and higher prevalence of PCOS, MENA patients had significantly lower oocyte yields than their European counterparts (P = 0.001). These findings suggest a more rapid decline in ovarian function in women of MENA descent.

[Metabolic and Reproductive Consequences of the Polycystic Ovary Syndrome (PCOS)]. / Metabolische und reproduktionsmedizinische Aspekte des Polyzystischen Ovarsyndroms (PCOS).

Polycystic ovarian syndrome represents the most common endocrine disease of women of reproductive age. Symptoms include metabolic, gynecologic and cosmetic features. Genetic factors seem to contribute to the disease, affecting not only women but also male relatives of patients with similar symptoms. Besides, lifestyle factors play a central role impacting clinical PCOS appearance. Following we present an overview of the syndrome, its epidemiology, metabolic and gynecological aspects, gender and genetic factors and its therapy.

Pluripotent Stem Cell-Derived In Vitro Gametogenesis and Synthetic Embryos-It Is Never Too Early for an Ethical Debate.

Recently, 2 branches of the wide area of synthetic biology-in vitro gametogenesis and synthetic embryo development-have gained considerable attention. Rodent induced pluripotent stem cells derived via reprogramming of somatic cells can in vitro be differentiated into gametes to produce fertile offspring. And even synthetic embryos with organ progenitors were generated ex utero entirely from murine pluripotent stem cells. The use of these approaches in basic research, which is rightfully accompanied by an ethical discussion, will allow hitherto unattainable insights into the processes of the beginning of life. There is a broad international consensus that currently the application of these technologies in human-assisted reproduction must be considered to be unsafe and unethical. However, newspaper headlines also addressed the putatively resulting paradigm shift in human reproduction and thereby raised expectations in patients. Due to unsolved biological and technological obstacles, most scientists do not anticipate translation of any of these approaches into human reproductive medicine, if ever, for the next 10 years. Still, whereas the usage of synthetic embryos for reproductive purposes should be banned, in the context of in vitro-derived human gametes it is not too early to initiate the evaluation of the ethical implications, which could still remain assuming all technological hurdles can ever be cleared.

Multipotent fetal stem cells in reproductive biology research.

Due to the limited accessibility of the in vivo situation, the scarcity of the human tissue, legal constraints, and ethical considerations, the underlying molecular mechanisms of disorders, such as preeclampsia, the pathological consequences of fetomaternal microchimerism, or infertility, are still not fully understood. And although substantial progress has already been made, the therapeutic strategies for reproductive system diseases are still facing limitations. In the recent years, it became more and more evident that stem cells are powerful tools for basic research in human reproduction and stem cell-based approaches moved into the center of endeavors to establish new clinical concepts. Multipotent fetal stem cells derived from the amniotic fluid, amniotic membrane, chorion leave, Wharton´s jelly, or placenta came to the fore because they are easy to acquire, are not associated with ethical concerns or covered by strict legal restrictions, and can be banked for autologous utilization later in life. Compared to adult stem cells, they exhibit a significantly higher differentiation potential and are much easier to propagate in vitro. Compared to pluripotent stem cells, they harbor less mutations, are not tumorigenic, and exhibit low immunogenicity. Studies on multipotent fetal stem cells can be invaluable to gain knowledge on the development of dysfunctional fetal cell types, to characterize the fetal stem cells migrating into the body of a pregnant woman in the context of fetomaternal microchimerism, and to obtain a more comprehensive picture of germ cell development in the course of in vitro differentiation experiments. The in vivo transplantation of fetal stem cells or their paracrine factors can mediate therapeutic effects in preeclampsia and can restore reproductive organ functions. Together with the use of fetal stem cell-derived gametes, such strategies could once help individuals, who do not develop functional gametes, to conceive genetically related children. Although there is still a long way to go, these developments regarding the usage of multipotent fetal stem cells in the clinic should continuously be accompanied by a wide and detailed ethical discussion.

The impact of regional origin on the incidence of gestational diabetes mellitus in a multiethnic European cohort.

Introduction: Women with migration background present specific challenges related to risk stratification and care of gestational diabetes mellitus (GDM). Therefore, this study aims to investigate the role of ethnic origin on the risk of developing GDM in a multiethnic European cohort. Methods: Pregnant women were included at a median gestational age of 12.9 weeks and assigned to the geographical regions of origin: Caucasian Europe (n = 731), Middle East and North Africa countries (MENA, n = 195), Asia (n = 127) and Sub-Saharan Africa (SSA, n = 48). At the time of recruitment maternal characteristics, glucometabolic parameters and dietary habits were assessed. An oral glucose tolerance test was performed in mid-gestation for GDM diagnosis. Results: Mothers with Caucasian ancestry were older and had higher blood pressure and an adverse lipoprotein profile as compared to non-Caucasian mothers, whereas non-Caucasian women (especially those from MENA countries) had a higher BMI and were more insulin resistant. Moreover, we found distinct dietary habits. Non-Caucasian mothers, especially those from MENA and Asian countries, had increased incidence of GDM as compared to the Caucasian population (OR 1.87, 95%CI 1.40 to 2.52, p < 0.001). Early gestational fasting glucose and insulin sensitivity were consistent risk factors across different ethnic populations, however, pregestational BMI was of particular importance in Asian mothers. Discussion: Prevalence of GDM was higher among women from MENA and Asian countries, who already showed adverse glucometabolic profiles at early gestation. Fasting glucose and early gestational insulin resistance (as well as higher BMI in women from Asia) were identified as important risk factors in Caucasian and non-Caucasian patients.