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.

An annotated human blastocyst dataset to benchmark deep learning architectures for in vitro fertilization.

Medical Assisted Reproduction proved its efficacy to treat the vast majority forms of infertility. One of the key procedures in this treatment is the selection and transfer of the embryo with the highest developmental potential. To assess this potential, clinical embryologists routinely work with static images (morphological assessment) or short video sequences (time-lapse annotation). Recently, Artificial Intelligence models were utilized to support the embryo selection procedure. Even though they have proven their great potential in different in vitro fertilization settings, there is still considerable room for improvement. To support the advancement of algorithms in this research field, we built a dataset consisting of static blastocyst images and additional annotations. As such, Gardner criteria annotations, depicting a morphological blastocyst rating scheme, and collected clinical parameters are provided. The presented dataset is intended to be used to train deep learning models on static morphological images to predict Gardner's criteria and clinical outcomes such as live birth. A benchmark of human expert's performance in annotating Gardner criteria is provided.

Antibody Response and Maternofetal Antibody Transfer in SARS-CoV-2-Positive Pregnant Women: A Multicenter Observational Study.

Introduction Awareness of SARS-CoV-2 infection in pregnant women and the potential risk for infection of their neonates is increasing. The aim of this study was to examine the immune status of affected women and evaluate the dynamics of placental antibody transfer. Materials and Methods The study included 176 women with SARS-CoV-2 infection during pregnancy who delivered between April 2020 and December 2021 at eight obstetric maternity sites. Demographic data, maternal and neonatal characteristics were summarized. Antibody testing for IgA and IgG in maternal blood sera and umbilical cord samples was evaluated and IgG transfer ratios were calculated. Values were related to the time of infection during pregnancy and birth. Results The percentage of IgG positive women increased from 29.0% (95% CI 23.8 - 37.8) at presentation with a positive PCR test result to 75.7% (95% CI 71.6 - 79.8), the percentage of IgG positive umbilical cord blood samples increased from 17.1% (95% CI 13.0 - 21.3) to 76.4% (95% CI 72.2 - 80.7) at more than six weeks after infection. Regression lines differed significantly between maternal and fetal IgG responses (p < 0.0001). Newborns react with a latency of about one week; umbilical cord blood antibody concentrations are highly correlated with maternal concentration levels (ρ = 0.8042; p < 0.0001). IgG transplacental transfer ratios were dependent on infection-to-birth interval. Two of the umbilical cord blood samples tested positive for IgA. Conclusions These findings confirm vertical SARS-CoV-2 transmission is rare; however, antibodies are transferred to the fetus soon after infection during pregnancy. Since transplacental antibody transfer might have a protective value for neonatal immunization this information may be helpful when counseling affected women.

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.

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.

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.

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 .

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.

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.

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.

Retrospective evaluation of established cut-off values for the sFlt-1/PlGF ratio for predicting imminent delivery in preeclampsia patients.

OBJECTIVE: The outcome of preeclampsia is difficult to predict. Laboratory markers such as soluble fms-like tyrosine kinase (sFlt-1) and placental growth factor (PlGF) are thought to be predictive factors. A 2012 study by Verlohren et al. evaluated cut-off values for assessing time to delivery in patients with elevated sFlt-1/PlGF ratios. The present study aimed to evaluate findings in patients with elevated sFlt-1/PlGF ratios who gave birth at Kepler University Hospital in Linz, Austria. The hypothesis was tested, that our patients show longer pregnancy duration despite elevated sFlt-1/PlGF ratios. STUDY DESIGN: This retrospective data analysis included all patients with sFlt-1/PlGF ratios above the established cut-off values between January 2014 and October 2017. Two groups were analyzed relative to gestational age and were matched with healthy controls: 24 + 0 to 33 + 6 gestational weeks, sFlt-1/PlGF ratio >655.2; and 34 + 0 to 36 + 6 gestational weeks, sFlt-1/PlGF ratio >201. MAIN OUTCOME MEASURES: sFlt-1/PlGF ratio and time to delivery correlation. RESULTS: In the <34-week group, 43.2% of the patients delivered beyond 48 h, with a mean sFlt-1/PlGF ratio of 885.06, showing a significantly lower sFlt-1/PlGF ratio than patients who delivered within 2 days (P = 0.04). In the >34-week group, 66.7% were still pregnant after 48 h, with a mean sFlt-1/PlGF ratio of 273.7. CONCLUSION: The sFlt-1/PlGF ratio appears to be a powerful tool for diagnosing and predicting preeclampsia. However, the data do not confirm the cut-off values described earlier, with longer pregnancy durations in this group of patients.

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.

Postpartum Galactostasis of the Vulva in a Case of Bilateral Lactating Ectopic Breast Tissue.

BACKGROUND: In the general population, the incidence of accessory breast tissue, a congenital malformation, is 1-5%. The most common site is the lower axilla. Detecting such tissue may be problematic, and accessory breasts below the umbilicus are extremely rare. CASE: This report describes the case of a 5-day postpartum 29-year-old woman, G2P2, with painful vulvar swelling 6 cm in diameter. The patient was diagnosed with polymastia in the vulva, without polythelia, with galactostasis due to suturing of a birth laceration covering an excretory duct. The sutures were removed, and the pain decreased. Breastfeeding was continued. CONCLUSION: Ectopic breast tissue is rare but should be taken into consideration in the differential diagnosis of a vulvar mass, especially postpartum in lactating women.

The Role of Endometrial Volume and Endometrial and Subendometrial Vascularization Parameters in a Frozen Embryo Transfer Cycle.

The role of three-dimensional power Doppler ultrasonography of the endometrium in assisted reproduction is still far from clear. In this retrospective cohort study, transvaginal three-dimensional power Doppler examinations were performed 30 min before frozen-thawed embryo transfer. After pregnancy tests, two cohorts were established: P (pregnant, n = 31) and NP (nonpregnant, n = 31). The study only included nullipara with no uterine abnormalities who were undergoing infertility treatment at the Department of Gynecology, Obstetrics and Gynecological Endocrinology, Kepler University Hospital, Linz, Austria. The main outcome measures were the vascularization flow index (VFI), flow index (FI), and vascularization index (VI) in the endometrium/subendometrium, assessed using Virtual Organ Computer-aided AnaLysis (VOCAL™), and the endometrial volume. A total of 62 patients were enrolled in the study, forming two cohorts (pregnant, P; nonpregnant, NP). There were no significant differences between the two cohorts with regard to demographic data, numbers of embryos transferred, or embryo grading, but there was a significant difference in endometrial volume (cohort P, 3.17 ± 0.84 mL; cohort NP, 2.36 ± 0.9 mL; P = 0.001) and the pregnancy rate rises with larger volume. No differences were observed in the vascularization parameters FI, VFI, and VI in the endometrium and subendometrium. In the cohort of pregnant patients, there were 26 (41.9%) live births, with 21 term deliveries (80.8%). The endometrial volume was larger in the cohort of pregnant patients. Measurements were performed 30 min before embryo transfer, and no differences were observed in vascularization parameters in the subendometrium and endometrium.

Good-quality blastocysts derived from vacuolized morulas show reduced viability.

OBJECTIVE: To study whether late spontaneous vacuolization on day 4 is an artefact or an alternate means of blastocele formation and to analyze its impact on pregnancy outcome and live birth. DESIGN: Prospective observational study. SETTING: University teaching hospital. PATIENT(S): A total of 424 patients who fulfilled inclusion criteria were subgrouped according to the spontaneous vacuolization on day 4: Group 1 had all morulas affected, group 2 showed no signs of vacuoles, and group 3 was mixed (some day 4 embryos had vacuoles and others did not). INTERVENTION(S): Screening for the presence of vacuoles on day 4 and fresh single-blastocyst transfer. MAIN OUTCOME MEASURE(S): Morula and blastocyst scoring, utilization rate, pregnancy and live birth rates. RESULT(S): Patients of group 1 had a reduced blastocyst formation rate on day 5 (P<.01) and significantly fewer good-quality blastocysts for usage (P<.05). In addition, pregnancy (P<.001) and live birth (P<.01) rate were significantly worse in group 1 compared with groups 2 and 3. CONCLUSION(S): Late onset of vacuolization around compaction stage is a negative predictor of blastocyst formation and outcome.

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.