Discovery of an unusually high number of de novo mutations in sperm of older men using duplex sequencing.

De novo mutations (DNMs) are important players in heritable diseases and evolution. Of particular interest are highly recurrent DNMs associated with congenital disorders that have been described as selfish mutations expanding in the male germline, thus becoming more frequent with age. Here, we have adapted duplex sequencing (DS), an ultradeep sequencing method that renders sequence information on both DNA strands; thus, one mutation can be reliably called in millions of sequenced bases. With DS, we examined ∼4.5 kb of the FGFR3 coding region in sperm DNA from older and younger donors. We identified sites with variant allele frequencies (VAFs) of 10-4 to 10-5, with an overall mutation frequency of the region of ∼6 × 10-7 Some of the substitutions are recurrent and are found at a higher VAF in older donors than in younger ones or are found exclusively in older donors. Also, older donors harbor more mutations associated with congenital disorders. Other mutations are present in both age groups, suggesting that these might result from a different mechanism (e.g., postzygotic mosaicism). We also observe that independent of age, the frequency and deleteriousness of the mutational spectra are more similar to COSMIC than to gnomAD variants. Our approach is an important strategy to identify mutations that could be associated with a gain of function of the receptor tyrosine kinase activity, with unexplored consequences in a society with delayed fatherhood.

Single day 14 serum hCG values allow prediction of viable pregnancy and are significantly higher in frozen as compared to fresh single blastocyst transfer.

PURPOSE: To evaluate if single serum human chorionic gonadotropin (hCG) level measurements are sufficient for pregnancy monitoring after single embryo transfer (sET) and to compare the hCG levels between fresh (FRET) and frozen embryo transfers (FET) in medically assisted reproduction. METHODS: This was a retrospective exploratory cohort study including all patients who met the inclusion criteria, who received a single FRET (n = 249) or FET (n = 410) of a day five blastocyst at the IVF clinic at the Johannes Kepler University Linz between 2011 and 2020. hCG levels were measured on day 14 after embryo transfer. Threshold values for the viability of pregnancies were determined using receiver operating characteristic (ROC) curves. RESULTS: Significantly higher hCG levels were found in those who received FET than in those who received FRET (1222.8 ± 946.7 mU/ml vs. 862.7 ± 572.9 mU/ml; p < 0.001). Optimal threshold values predicting a viable pregnancy were 368.5 mU/ml and 523 mU/ml in the FRET and FET groups, respectively. CONCLUSIONS: After FET, higher hCG values after 14 days of embryo transfer must be considered in pregnancy monitoring. Additionally, a single threshold hCG value seems to be sufficient for determining pregnancy viability. To exclude ectopic pregnancies, subsequent ultrasound examination is a mandatory requirement.

Double ionophore application in cases with previous failed/low fertilization or poor embryo development.

RESEARCH QUESTION: Does a double ionophore application improve the outcome of cycles in which single ionophore application was unsuccessful? DESIGN: This retrospective intervention study (duration 4.5 years) included 79 patients with suspected chronic failed oocyte activation (<30% fertilizations) and/or poor embryo development (developmental arrest, 24 h developmental delay, blastulation rate <15%) in both preceding cycles, the first without ionophore and the second with single ionophore treatment. Within the study period, all patients with failed ionophore treatments (single applications of ready-to-use calcimycin for 15 min) were offered an adapted protocol in the subsequent cycle (study cycle) in which the same ionophore was applied twice (separated by 30 min). Tests for paired data (control and study cycle) were used to reduce the effect of confounders. RESULTS: The overall fertilization rate did not differ between the study and control cycles. Cleavage (P = 0.020) and blastocyst formation (P = 0.018) rates improved significantly in the study cycles. Implantation (P = 0.001), biochemical (P < 0.001) and clinical pregnancy (P < 0.001) rates were also significantly higher in the study cycles. The study cycles resulted in 29 live births and all 32 babies born were healthy. CONCLUSIONS: This study suggests that double ionophore application may improve blastocyst formation and clinical pregnancy rates in cases of failed single ionophore treatment, irrespective of whether the ionophore was used to overcome fertilization failure or poor embryo development. Fertilization rate was only increased in cases with a history of fertilization failure. Because single ionophore treatment was used in only one previous cycle it cannot be ruled out that some improvement in clinical outcomes would also have been achieved by using single instead of double ionophore treatment again in the subsequent attempt.

Preoperative application of the Enzian classification for endometriosis (The cEnzian Study): A prospective international multicenter study.

OBJECTIVE: To assess the diagnostic performance of preoperative application of the Enzian classification (cEnzian) using surgical findings as reference standard. DESIGN: A prospective international non-interventional study. SETTING: Twelve endometriosis centres in four European countries (Austria, Germany, Switzerland and Czech Republic). POPULATION: 1062 women with endometriosis surgery. METHODS: Extent of endometriosis was preoperatively classified using the cEnzian classification based on gynaecological examination and/or transvaginal ultrasound (TVS) and/or magnetic resonance imaging (MRI). After subsequent surgery, the surgeon classified the intraoperative findings using the Enzian classification. MAIN OUTCOME MEASURES: Sensitivity, specificity, PPV, NPV, LR+ , LR- and accuracy were calculated. Conditional frequencies of intraoperative Enzian codings and the corresponding 95% confidence intervals were computed for each preoperative coding and visualised in plots. RESULTS: Although overall consistency of cEnzian and Enzian was poor (35.14%, 95% confidence interval 32.26-38.03), high specificities and negative predictive values (NPVs) of the cEnzian compartments could be demonstrated. Looking at the individual parts of the Enzian classification, the poorest diagnostic performance was detected for compartment B and the highest PPVs were found for category 3 lesions (>3 cm), independent of the compartment. CONCLUSIONS: Using the Enzian classification in a non-invasive setting is a useful tool providing us with an 'at a glance' summary of the diagnostic workup regarding deep endometriosis with high specificities and NPVs. An attempt to merge the two new endometriosis classification systems (#Enzian and AAGL 2021) seems reasonable taking into consideration the respective advantages of each other.

Bleeding pattern after medical management of early pregnancy loss with mifepristone-misoprostol and its prognostic value: a prospective observational cohort study.

PURPOSE: To improve counseling of women by reporting bleeding characteristics at home after medical management of an early pregnancy loss (EPL) with mifepristone and misoprostol, and to evaluate occurring bleeding patterns as a prognostic tool. METHODS: This prospective two-center observational cohort study enrolled 197 women who presented with an EPL (embryonic or anembryonic miscarriage) from December 2017 to April 2019 and chose a home-based medical management with 200 mg mifepristone and 800 mcg misoprostol. From the day of mifepristone intake, the strength of vaginal bleeding was recorded daily for 2 weeks by the patient herself using a diary sheet. Treatment success was defined as no histologically confirmed retained products of conception (RPOC) within 3 months. After considering all drop-out criteria, 154 women were included in the analysis. RESULTS: 40.0% of patients (95% CI 30.4-49.6) already reported bleeding onset in the time period between the intake of mifepristone and misoprostol. The median duration of vaginal bleeding including spotting was 13 days. The chance of RPOC was about sixfold (OR 6.06, 95% CI 2.15-17.10) in the group of persistent bleeding after 2 weeks compared to the group with a terminated bleeding at that time. Exploratory regression analysis indicated association of higher serum levels of leukocytes at treatment start with RPOC (p = 0.013). CONCLUSIONS: Terminated bleeding after 2 weeks is a useful indicator for successful medical induction of EPL. Women undergoing medical treatment with mifepristone must be informed about the high frequency of bleeding onset before misoprostol intake. CLINICAL TRIAL REGISTRATION: DRKS-German Clinical Trials Register, ID: DRKS00013515, registration date 05.12.2017. http://www.drks.de/DRKS00013515 .

The hare and the tortoise: extreme mitotic rates and how these affect live birth.

RESEARCH QUESTION: Is live birth of patients with excessive slow (no blastocyst on day 5) and fast mitotic rate (full blastocyst development on day 4) comparable to a matched control standard (blastocyst formation on day 5)? DESIGN: In this retrospective matched (age and anti-Müllerian hormone [AMH]) case-control study rates of fertilization, blastulation, implantation, clinical pregnancy and live birth were compared in couples with male factor indication, prolonged embryo culture and fresh single morula and blastocyst transfer. RESULTS: The rates of implantation, clinical pregnancy and live birth in the slow-developing group were significantly (P < 0.001) lower (17.6%, 13.7%, and 11.8%, respectively) compared with the fast (58.5%, 52.5%, 47.5%) and normal growing counterparts (51.5%, 42.6%, 39.6%). No differences in neonatal outcome could be observed between the three groups. Sex ratio in the fast-growing group was not different from the other cohorts. CONCLUSIONS: Extremely slow development, as assessed by the absence of blastulation on day 5, is a negative predictor of pregnancy and live birth. In contrast, the fear that extremely fast-growing embryos may represent an aneuploid cohort of embryos is unsubstantiated. Day-4 full blastocysts can preferentially be considered for transfer.

Ionophore application for artificial oocyte activation and its potential effect on morphokinetics: a sibling oocyte study.

PURPOSE: To evaluate whether ionophore application at the oocyte stage changes the morphokinetics of the associated embryos in cases of artificial oocyte activation. METHODS: In a prospective sibling oocyte approach, 78 ICSI patients with suspected fertilization problems had half of their MII-oocytes treated with a ready-to-use ionophore (calcimycin) immediately following ICSI (study group). Untreated ICSI eggs served as the control group. Primary analyses focused on morphokinetic behavior and the presence of irregular cleavages. The rates of fertilization, utilization, pregnancy, and live birth rate were also evaluated. RESULTS: Ionophore-treated oocytes showed a significantly earlier formation of pronuclei (t2PNa) and a better synchronized third cell cycle (s3) (P < .05). The rate of irregular cleavage was unaffected (P > .05). Ionophore treatment significantly improved the overall rates of fertilization (P < .01) and blastocyst utilization (P < .05). CONCLUSION: Ionophore application does not negatively affect cleavage timing nor is it associated with irregular cleavage.

Retrospective evaluation of attempted vaginal deliveries in dichorionic twin pregnancies.

PURPOSE: Numbers of planned cesarean deliveries are increasing in twin pregnancies, despite the lack of evidence for this approach, and the second twin is thought to be at risk for a poorer outcome. The aim of this study was to examine whether twins have a poorer outcome if an attempted vaginal delivery is changed to a cesarean section or combined delivery. METHODS: This retrospective data analysis included all women with dichorionic twin pregnancies attempting vaginal delivery over a 10-year period. Outcome parameters for the first and second twins relative to their mode of birth were compared. A correlation model between the interdelivery time interval and Apgar scores was calculated. Subgroup analyses assessing the birth mode of the first and second twins were conducted. RESULTS: A total of 248 women were enrolled in the study. The second twins had significantly lower values for outcome parameters, such as umbilical artery cord pH and Apgar scores in comparison with the first twins (P < 0.01). The subgroup analysis of birth modes in first and second twins showed a significantly poorer outcome in the cesarean section and combined delivery group (P < 0.05). The interdelivery time interval was significantly longer in the second twin cesarean section group (P < 0.01). There was no significant correlation between the interdelivery time intervals and Apgar scores (P > 0.05). CONCLUSION: Although outcome parameters were significantly lower in second twins and twins born via secondary cesarean section, the clinical relevance of this appears to be negligible.

Time-lapse imaging of cytoplasmic strings at the blastocyst stage suggests their association with spontaneous blastocoel collapse.

RESEARCH QUESTION: To study the origin and temporal behaviour of cytoplasmic strings spanning the blastocoel (main objective) and their influence on treatment outcome (secondary objective). DESIGN: This retrospective analysis of prospectively collected data was set up in a university medical centre. Patients who either underwent fresh (n = 95) or vitrified-warmed (n = 55) single blastocyst transfer were included. Time-lapse sequences of in-vitro developed blastocysts were screened for the presence of cytoplasmic strings. Pregnancies in string-positive and string-negative transfers were followed up to live birth. RESULTS: A total of 387 blastocysts were obtained in the fresh cycles of 100 patients, corresponding to a blastocyst formation rate of 62.4%. Cytoplasmic strings were first detected around full stage (108.5 ± 6.4 h) in 170 blastocysts (43.9%). The number of strings varied (range: 1-7) and the duration of visibility was 5.2 ± 3.5 h. The occurrence of cytoplasmic strings was significantly associated with the presence of blastocoelic collapses (P < 0.001) but not with any of the annotated morphokinetic parameters. Live birth and neonatal outcome were the same for both string-positive and string-negative pregnancies. Moreover, collapses did not affect treatment outcome. CONCLUSION: Time-lapse analysis of cytoplasmic strings at the blastocyst stage revealed that this morphological feature was not a negative predictor as previously reported. Although physiologically normal, at least some of the cytoplasmic strings are an artefact, possibly associated with blastocoelic collapses.

Oocyte competence in in vitro fertilization and intracytoplasmic sperm injection patients suffering from endometriosis and its possible association with subsequent treatment outcome: a matched case-control study.

INTRODUCTION: Endometriosis affects up to 15% of women of reproductive age. There is an obvious lack of studies dealing with morphological parameters of oocyte morphology in endometriosis patients in assisted reproduction. One aim of the study is to describe oocyte morphology in patients undergoing intracytoplasmic sperm injection suffering from endometriosis. In addition, the impact of endometriosis on in vitro fertilization results is analyzed. Both in vitro fertilization and intracytoplasmic sperm injection patients are then matched with an endometriosis-free control group for highlighting the possible association of endometriosis with pregnancy outcome. MATERIAL AND METHODS: Oocyte morphology of endometriosis patients was assessed in two groups. Both study group and control group consisted of 129 in vitro fertilization/intracytoplasmic sperm injection cycles each. Patients were matched according to anti-Müllerian hormone, female age, previous treatment cycles, and method of fertilization. Endometriosis was graded according to the revised American Society for Reproductive Medicine guidelines of 1997. RESULTS: Patients with endometriosis had a significantly lower rate of mature oocytes (p < 0.03) and morphologically normal oocytes (p < 0.001). In particular, brownish oocytes (p < 0.009; stage I-IV) and the presence of refractile bodies (p < 0.001; stage IV) were found to be increased. Endometriosis stage IV was associated with significantly worse-quality oocytes than stages I-III (p < 0.01). Fertilization was significantly reduced in conventional in vitro fertilization but not in intracytoplasmic sperm injection (p < 0.03). This was due to lower fertilization rates in stage III-IV endometriosis compared with stage I-II (p < 0.04). No difference was observed with respect to rates of implantation, clinical pregnancy, miscarriage, live birth, and malformation. CONCLUSIONS: Endometriosis patients, in particular those with severe endometriosis, present lower-quality oocytes. Once fertilized, no impairment of further preimplantation embryo development and pregnancy outcome right up to healthy live birth rate has to be expected.

Time-lapse imaging provides further evidence that planar arrangement of blastomeres is highly abnormal.

PURPOSE: Recently, guidelines on the annotation of dynamic human embryo monitoring recommended screening for the presence of planar blastomere arrangement at the 4-cell stage. This observational study was set up in order to analyze whether developmental kinetics of planar human embryos are different from tetrahedral ones. METHODS: Therefore, embryos of 115 consecutive ICSI patients (showing 32 planar and 554 tetrahedral embryos) were cultured in a new time-lapse system (Miri TL) and their embryos were annotated for morphokinetic development and screened for irregular cleavages and morphological dysmorphisms. RESULTS: Significantly less planar embryos reached blastocyst stage and showed worse quality as compared to regular tetrahedral embryos. The rate of bi- and/or multinucleation was also significantly higher in the affected group. Irregular cleavages, particularly embryo rolling, were more often seen in planar embryos. Morphokinetics between planar and tetrahedral were distinguishable up to 4-cell stage (t2-t4), thereafter the observed delay in planar embryos (t8) was more likely the result of a higher rate of arrested embryos in the planar group. CONCLUSIONS: Planar embryos are associated with both a significant increase in irregular cleavage as well as a delay in preimplantation development. This indicates that planar embryos are rather abnormal and should only be considered for transfer if no other embryos are available.

Quantitative and qualitative trophectoderm grading allows for prediction of live birth and gender.

PURPOSE: Prolonged in vitro culture is thought to affect pre- and postnatal development of the embryo. This prospective study was set up to determine whether quality/size of inner cell mass (ICM) (from which the fetus ultimately develops) and trophectoderm (TE) (from which the placenta ultimately develops) is reflected in birth and placental weight, healthy live-birth rate, and gender after fresh and frozen single blastocyst transfer. METHODS: In 225 patients, qualitative scoring of blastocysts was done according to the criteria expansion, ICM, and TE appearance. In parallel, all three parameters were quantified semi-automatically. RESULTS: TE quality and cell number were the only parameters that predicted treatment outcome. In detail, pregnancies that continued on to a live birth could be distinguished from those pregnancies that aborted on the basis of TE grade and cell number. Male blastocysts had a 2.53 higher chance of showing TE of quality A compared to female ones. There was no correlation between the appearance of both cell lineages and birth or placental weight, respectively. CONCLUSIONS: The presented correlation of TE with outcome indicates that TE scoring could replace ICM scoring in terms of priority. This would automatically require a rethinking process in terms of blastocyst selection and cryopreservation strategy.

The rASRM score and the Enzian classification for endometriosis: their strengths and weaknesses.

The revised American Society for Reproductive Medicine (rASRM) score is currently the best-known classification of endometriosis and is the one most widely used throughout the world. It is relatively easy to use, but it does not take into account the involvement of retroperitoneal structures with deeply infiltrating endometriosis. For this reason, the Enzian classification was developed as a supplement to the rASRM score, in order to provide a morphologically descriptive classification of deeply infiltrating endometriosis. The Enzian classification currently has a poor level of international acceptance and is mainly used in the German-speaking countries. It was revised in 2011 and is now also easier to use. This article describes the strengths and weaknesses of the rASRM score and the Enzian classification and their common potential for classifying endometriosis.

Enzian classification: does it correlate with clinical symptoms and the rASRM score?

OBJECTIVE: To assess the extent to which the Enzian classification correlates with the revised American Society for Reproductive Medicine (rASRM) score and clinical symptoms in women with deeply infiltrating endometriosis. DESIGN: Retrospective cohort study (Canadian Task Force classification II-2). SETTING: Endometriosis competence center specializing in minimally invasive surgery. PATIENTS: Between 1 January 2009 and 31 December 2011, a total of 194 women underwent surgery due to deeply infiltrating endometriosis. After histological confirmation, they were classified using the rASRM and Enzian systems. Clinical symptoms were recorded preoperatively. INTERVENTIONS: Operative laparoscopy to treat endometriosis. MAIN OUTCOME MEASURES AND RESULTS: A clear correlation was seen between grades of severity in the rASRM score and the Enzian classification (p < 0.001). In addition, the rASRM severity grade and clinical symptoms correlated with the locations in the Enzian classification in relation to deeply infiltrating endometriosis. Pain and dysmenorrhea correlated strongly (p = 0.002, p < 0.001) with the severity grade in the Enzian classification. CONCLUSIONS: Deeply infiltrating endometriosis is well characterized using the Enzian classification as a supplement to the rASRM score. There is also a clear correlation between the rASRM and Enzian classifications, because of the way in which the disease crosses morphological boundaries. The locations in the Enzian classification correlate partially with clinical symptoms, and the classification's severity grades correlate substantially with pain and dysmenorrhea. In view of these clinical results, use of the Enzian classification can be recommended as a supplement to the rASRM score for detailed description of endometriosis.

Allen-Masters syndrome: do the classic risk factors also apply in patients with endometriosis?

AIM: To evaluate whether the classic risk factors for Allen-Masters syndrome, such as body mass index (BMI) and spontaneous births, are in fact responsible for the condition in patients with endometriosis. METHODS: A total of 26 women who underwent a laparoscopic procedure due to chronic pelvic pain, Allen-Masters syndrome and endometriosis from 2009-2011 were enrolled in this study from an endometriosis competence center specializing in minimally invasive surgery. This was a retrospective cohort study (Canadian Task Force classification II-2). RESULTS: Only eight of the 26 patients (30.77%; 95% confidence interval [CI], 14.33-51.79%) had the classic risk factors (BMI >25 kg/m(2) and/or at least one spontaneous birth). The mean age in the study group was 32.08 years (SD ± 5.45). The patients had a mean BMI (kg/m(2) ) of 19.61 (SD ± 3.07). The means for the patients' clinical data were 0.88 (SD ± 1.53) pregnancies, 0.81 (SD ± 1.23) for parity, 0.27 (SD ± 0.60) for cesareans and 0.54 (SD ± 0.99) for spontaneous births. The revised American Society for Reproductive Medicine (rASRM) stage showed a median of grade III. The left side of the posterior compartment was more often affected (73.1% of cases) than the right side in all patients. CONCLUSION: In all, 69.23% of the cases (95% CI, 48.21-85.67%) were not explained by the classic risk factors. In view of the absence of other diseases and otherwise unremarkable parameters, we consider mechanical damage of the lesser pelvis not to be solely responsible for AMS. Further observations in patients with AMS, with and without endometriosis, may be able to contribute to research into the actual etiology of the condition.

Costs of endometriosis in Austria: a survey of direct and indirect costs.

PURPOSE: The literature includes a wealth of medical data on endometriosis, but the economic significance of the condition has so far been neglected. An analysis of hospital costs for endometriosis in Austria was, therefore, carried out for economic purposes. METHODS: Seventy-three patients with endometriosis were included in the study. A bottom-up approach was used to collect data on the average hospital costs of an endometriosis patient over a time period of 1 year. In addition, a prevalence approach was used that allows subsequent estimation of the total costs of endometriosis for the health-care system in Austria for that period. Retrospective questionnaire survey was conducted. RESULTS: The average annual costs of one case of endometriosis are 7,712, with 5,605.55 attributable to direct costs and 2,106.34 to indirect costs. This indicates an overall economic burden of 328 million. In-patient care (45 %) and loss of productivity (27 %) were identified as the major cost factors. The patients themselves pay for 13 % of the costs (through out-of-pocket payments). CONCLUSIONS: This study impressively demonstrates the financial burden on the economy and on each individually affected patient caused by the disease of endometriosis. The massive consumption of resources represents a high level of usage of the medical services provided. The question arises as to whether more timely diagnosis, followed by better-targeted treatment, might have the potential to reduce these costs. The overall economic burden of endometriosis in Austria is currently comparable with that of Parkinson's disease.

Efficacy of the revised Enzian classification: a retrospective analysis. Does the revised Enzian classification solve the problem of duplicate classification in rASRM and Enzian?

PURPOSE: The most widely accepted classification for endometriosis is the Revised American Society for Reproductive Medicine (rASRM) system, but this does not take deeply infiltrating endometriosis (DIE) into account. The Enzian classification enables clinicians to classify DIE. Due to complexity and partial overlap with rASRM, it was revised for a second time in February 2011. Using both the systems to classify lesions would be inappropriate, as they refer to different locations. The aim of this study was to analyze whether the revised Enzian classification is easier to use and avoids duplicate classifications. METHODS: Retrospective study of 460 women admitted for endometriosis. RESULTS: One hundred and eighty-seven of 460 patients (41 %) had histologically confirmed DIE based on the revised Enzian classification. Further classification of these 187 patients using Enzian revealed 270 retroperitoneal lesions, as some patients had several DIE-type lesions simultaneously: 66 in compartment A (rectovaginal septum, vagina), 112 in compartment B (sacrouterine ligaments, pelvic wall), 58 in compartment C (bowel), 15 with adenomyosis uteri, 7 with bladder involvement, 8 with intrinsic involvement of the ureter, and 4 with bowel involvement. All 270 lesions were classified using Enzian alone and not with the rASRM score. There were no duplicate classifications (rASRM and Enzian). CONCLUSIONS: The revised Enzian classification is an excellent complement to the rASRM score for morphological description of DIE.