Vitamin D and anti-Müllerian hormone concentration in human follicular fluid individually aspirated from all patient follicles.

OBJECTIVE: The main aim of this prospective study was to investigate the relationship between intrafollicular vitamin D and anti-Müllerian hormone (AMH) concentration and its impact on oocyte quality and developmental competence. METHODS: The analysis was performed on 208 follicular fluid (FF) samples obtained from 33 patients undergoing ovarian stimulation as part of in vitro fertilization (IVF) treatment that included intracytoplasmic sperm injection. RESULTS: Our study shows that vitamin D concentration in FF varies according to the developmental stage of the oocyte and corelates with embryo development status on day 3, while AMH concentration in FF is not correlated with the developmental potential of an oocyte. We demonstrated that the levels of vitamin D and AMH were higher in FF than in serum. Moreover we showed that AMH and vitamin D levels were positively correlated in FF but not in serum. CONCLUSION: FF-AMH levels do not appear to be a suitable as noninvasive test of the developmental potential of an oocyte, while FF-vitamin D level can be used to evaluate whether embryos obtained from particular oocytes have potential of reaching the third day of culture. However, our results encourage further research to be carried out on a larger number of patients and testing additional components found in FF such as androgens.

Effect of next-generation sequencing in preimplantation genetic testing on live birth ratio.

The present study analysed live birth ratios in frozen embryo transfer (FET) cycles where embryo ploidy status was determined with preimplantation genetic testing (PGT) using next-generation sequencing (NGS). PGT was performed on trophectoderm cells biopsied at the blastocyst stage. The present prospective cohort study included 112 women undergoing frozen embryo transfer, with NGS PGT. The control group consisted of 85 patients who underwent the IVF procedure with FET planned for a subsequent cycle. The live birth rate per cycle was higher by ~18.5 percentage points in the investigated compared with control group (42.0% vs 23.5% respectively; P=0.012). The differences between the study and control groups were also significant for clinical pregnancy (42.0% vs 23.5% respectively; P=0.012), implantation (41.2% vs 22.2% respectively; P=0.001) and pregnancy loss rates (9.6% vs 28.6% respectively; P=0.027). The results show that PGT NGS is a useful method for embryo selection and it may be implemented in routine clinical practice with propitious results.

Clinical utility of different anti-Müllerian hormone - AMH assays for the purpose of pregnancy prediction.

PURPOSE: Comparison of outcomes of IVF cycles where the AMH levels was measured with five different AMH kits: Immunotech (IOT), Beckman Coulter II Gen. RUO, Beckman Coulter II Gen. IVD (BC II IVD), Ansh Labs ultrasensitive (Ansh) and the automated Elecsys Roche assay. METHODS: Retrospective analysis of clinical data for 3693 cycles. RESULTS: In women < 35 years with low (<0.6 ng/ml) and high (>1.4 ng/ml) AMH concentrations, and in those > 39 years with medium (≥0.6 and ≤1.4 ng/ml) and high AMH concentrations the clinical pregnancy rate differed significantly among groups of patients whose AMH level was measured with different kits. In those subgroups, the highest rates were recorded for the BC II IVD and Ansh groups, while the lowest in the IOT group. AMH concentrations differed significantly between different kits in all age groups (the highest in each age group was for the IOT kit and the lowest for BC II IVD). AMH correlates positively with antral follicle count, MII and number of oocytes retrieved. CONCLUSIONS: This study demonstrated that we could expect very different pregnancy rates with the same AMH results depending on the AMH kit used. That would means, different values of AMH could similarly lead to misleading clinical decisions in IVF.

New AMH assay allows rapid point of care measurements of ovarian reserve.

In this study, we compare two commercial automated immunoassays used to evaluate serum anti-Müllerian hormone (AMH) levels as a prognostic value for ovarian response and pregnancy outcome in assisted reproductive technology cycles. Serum AMH was measured for 193 women. We performed a simultaneous measurement in serum AMH with the two alternative kits VIDAS® and Elecsys® AMH assay. For all women undergoing in vitro fertilization cycle, we collected data on their antral follicle count (AFC) and numbers of retrieved cumulus oocyte complexes (OC) and metaphase II oocytes and pregnancy outcome. The AMH values provided by VIDAS® were correlated with the values obtained with Elecsys® (0.977 for fresh and 0.971 for the frozen samples). For both assays AMH exhibited a moderate positive correlation with AFC, OC and MII oocytes (0.612, 0.674, 0.605 for VIDAS® and 0.570, 0.617, 0.530 for Elecsys®, respectively). AMH prediction of biochemical and clinical pregnancy was similar. The present results suggest that the VIDAS® AMH assay is broadly comparable to the Elecsys-AMH assay in terms of technical performance for clinical or epidemiological use. Both automated assays performed in a similar way and the choice of assay can be made depending on the technical configuration of each laboratory.

Comparison of the second-generation Beckman Coulter IVD and first-generation AnshLabs ELISA assays for anti-Müllerian hormone in patients undergoing IVF treatment.

OBJECTIVES: Ovarian reserve is the main factor influencing the efficacy of infertility treatment. Currently the anti- Müllerian hormone is the main indicator of the ovarian reserve and has a wide spectrum of clinical importance. It achieved a high clinical value right after the introduction of the first commercial AMH assays in 2005. Lack further research and development of the tests and monopoly on their production have led to a significant reduction of their quality resulting in lowered veracity and usefulness. Therefore, we searched for an alternative to the Beckman Coulter assay. The objective of the study was to draw a comparison between the commonly used second-generation assay by Beckman Coulter and the ultra-sensitive first-generation assay by AnshLabs. MATERIALS AND METHODS: Serum samples (n=520) were collected from female patients undergoing routine AMH evaluation before entering an IVF program. We chose samples of patients with the lowest correlation between the AMH serum level and response to stimulation. The AMH serum levels of the patients were examined using two AMH tests, the second-generation assay by Beckman Coulter and the first-generation assay by AnshLabs. Precision and accuracy of both methods were determined and the results of AMH serum levels of 130 patients were correlated with the number of: antral follicles (AFC), follicles after stimulation, and the obtained cumulus cells. RESULTS: Both precision and accuracy of the compared methods were highly satisfactory. The coefficients of variation obtained in the study conducted on two different levels of control material were lower than 12% and the load did not exceed 9%. The study proved that both of the methods yielded comparable results. The coefficient of variation between the first-generation and the second-generation AMH assays was 0.871. CONCLUSION: Both methods might be applied in the evaluation of the ovarian reserve. The first- and second-generation assays show comparable correlation with the clinical effects of stimulation, however it seems that first-generation assays are a better alternative to the unstable second-generation kits. The results from the first-generation assays are distributed on a wider range, which facilitates clinical interpretation.

IMSI-Guidelines for Sperm Quality Assessment.

Intracytoplasmic sperm injection (ICSI) is a widely used and accepted treatment of choice for oocyte fertilization. However, the quality of sperm selection depends on the accurate visualization of the morphology, which can be achieved with a high image resolution. We aim to correct the conviction, shown in a myriad of publications, that an ultra-high magnification in the range of 6000×-10,000× can be achieved with an optical microscope. The goal of observing sperm under the microscope is not to simply get a larger image, but rather to obtain more detail-therefore, we indicate that the optical system's resolution is what should be primarily considered. We provide specific microscope system setup recommendations sufficient for most clinical cases that are based on our experience showing that the optical resolution of 0.5 µm allows appropriate visualization of sperm defects. Last but not least, we suggest that mixed research results regarding the clinical value of IMSI, comparing to ICSI, can stem from a lack of standardization of microscopy techniques used for both ICSI and IMSI.

Follicular fat-soluble vitamins as markers of oocyte competency.

The main aim of this prospective study was to investigate the effect of the concentration of fat-soluble vitamins A, D, E and K in individual follicles on oocyte quality and developmental competence. The analysis was performed on 313 follicular fluid (FF) samples from 50 patients undergoing ovarian stimulation with intracytoplasmic sperm injection. We demonstrated that the mean concentration of individual vitamins in FF correlated with their level in serum (p < 0.0001). The levels of vitamin D in FF were higher than in serum, while the opposite was observed for other analyzed vitamins. We did not observe a correlation between FF vitamin D concentration with fertilization success. However, we observed its association with embryo development status on day 3. Moreover, we showed a statistically significant negative correlation between the mean day 5 embryo score and the concentration of vitamin D in serum (rS = -0.68 p = 0.01) and follicular fluid (rS = -0.71 p = 0.01). Our study showed that FF concentration of vitamin A and E was helpful in the prediction of fertilization success of each individual oocyte. Moreover, vitamin A and E concentrations in FF were associated with status of embryo development on the third day of culture. Vitamin A was also associated with the embryo quality on day 2 and the embryo development status on day 5 after fertilization. In conclusion, a combination of FF vitamin analysis and routine morphological assessment could allow for a more accurate and sensitive method of determining embryonic developmental competence and enable the selection of a better embryo to transfer and perhaps translating into an increased chance of pregnancy.Abbreviations: in vitro fertilization: IVF; anti-Mullerian hormone: AMH; follicular fluid: FF; intracytoplasmic sperm injection: ICSI; top quality: TQ; vitamin D binding globulin level: VDBP; assisted reproductive technology: ART.

Do serum androgens influence blastocysts ploidy in karyotypically normal women?

The aim of the study was to determine if serum testosterone (T) and dehydroepiandrosterone (DHEAS) levels are a factor in determining increased risk for embryonic aneuploidy in karyotypically normal women undergoing in vitro fertilization (IVF) and preimplantation genetic testing screening for aneuploidy (PGT-A). This is a retrospective cohort study of IVF cycles with PGT-A performed during 2015-2016. A total of 256 cycles with 725 embryos were initially considered for inclusion. A total of 208 cycles and 595 embryos determined to be either euploid or aneuploid were included in the analysis. The mean age of women was 37.4 ± 4.4 years. There were 193 (32.44%) euploid, and 338 (56.81%) aneuploid blastocysts. Sixty-four (10.76%) had 'no diagnosis' after PGT-A. The 32 embryos with 'no diagnosis' after first PGT-A were biopsied again and after the second analysis, 7 were found to be euploid and 3 aneuploid. The remaining 32 embryos were not reanalyzed due to the lack of patients' consent for the second biopsy. The relationship between embryo ploidy and levels of serum testosterone and dehydroepiandrosterone sulfate was assessed using ordinal multivariable regression analysis. The model, adjusted for both anti-Mullerian hormone (AMH) and age, showed no association between ploidy status and serum levels of the two hormones. We concluded that the serum levels of testosterone and DHEAS do not influence embryo ploidy in karyotypically normal women undergoing IVF. Abbreviations: T: testosterone; DHEAS: dehydroepiandrosterone; IVF: in vitro fertilization; PGT-A: preimplantation genetic testing screening for aneuploidy; AMH: anti-Mullerian hormone; FSH: follicle-stimulating hormone; LH: luteinizing hormone; E2: oestradiol; P: progesterone.

Next generation sequencing for preimplantation genetic testing of blastocysts aneuploidies in women of different ages.

Most of the current preimplantation genetic screening of aneuploidies tests are based on the low quality and low density comparative genomic hybridization arrays. The results are based on fewer than 2,700 probes. Our main outcome was the association of aneuploidy rates and the women's age. Between August-December 2013, 198 blastocysts from women (mean age 36.3+-4.6) undergoing in vitro fertilization underwent routine trophectoderm biopsy. NGS was performed on Ion Torrent PGM (Life Technologies). The results were analyzed in five age groups (<31, 31-35, 36-38, 39-40 and >40). 85 blastocysts were normal according to NGS results. The results in the investigated groups were (% of normal blastocyst in each group): <31 (41.9%), 31-35 (47.6%), 36-38 (47.8%), 39-40 (37.7%) and >40 (38.5%). Our study suggests that NGS PGD is applicable for routine preimplantation genetic testing. It allows also for easy customization of the procedure for each individual patient making personalized diagnostics a reality.

The role of vitamin D in reproductive dysfunction in women - a systematic review.

Vitamin D is essential for the proper functioning of the human body. There is also evidence of its strong association with fertility problems in women. This review aims to evaluate the relationship between vitamin D and diseases affecting women's fertility (polycystic ovarian syndrome (PCOS), uterine leiomyomas and endometriosis), and in vitro fertilization (IVF) outcome. A systematic review of the literature was conducted in Scopus and PubMed for relevant English language publications since 1989. Vitamin D influences the functioning of the reproductive system in women and has been associated with PCOS, uterine leiomyomas, endometriosis and in vitro fertilization (IVF) outcome. However, further studies on larger groups of patients are needed to establish what role vitamin D plays in the treatment of female infertility.

Sperm parameters and DNA fragmentation of balanced chromosomal rearrangements carriers.

INTRODUCTION: Somatic chromosomal rearrangements that occur in infertile males are thought to be one of the major genetic factors influencing male infertility. The objective of this retrospective study was to evaluate sperm parameters in a group of patients with balanced translocations. MATERIAL AND METHODS: We analyzed semen of 84 balanced somatic translocation carriers [35 Robertsonian translocation (RT group) and 49 reciprocal translocation (RCT group)] and 57 men with normal karyotype (control group). Semen samples were evaluated for sperm concentration, its motion parameters and vitality, round cell number (CASA) and DNA fragmentation index (TUNEL). Cytogenetic evaluation was also performed for each study participant. RESULTS: Sperm concentrations were lower when comparing the RT group to the control (p < 0.001) and RCT groups (p < 0.05). Occurrence of abnormal sperm concentration was more common among RT carriers (74.3%) than in the other groups (42.9% in RCT group and 28.1% in control group). The sperm progressive motility and vitality in RT carriers (21.53% and 62.17%) were lower than in control group (39.77% and 77.47%, p < 0.001, respectively) and RCT carriers (31.47% and 76.17%, p < 0.001, respectively). The RCT carriers and the control group did not differ in regard to sperm concentration, progressive sperm motility, motility grade D and sperm vitality. There were no significant differences in DNA fragmentation in carriers of both studied structural chro­mosomal rearrangements in comparison to subjects with normal karyotype. CONCLUSIONS: RT carriers had significantly lower semen parameters in comparison to not only the subjects with normal karyotypes but also the RCT carriers.

First Pregnancy, Somatic and Psychological Status of a 4-Year-Old Child Born following Annexin V TESA Sperm Separation.

Introduction Sperm DNA integrity is a crucial paternal factor affecting fertilization and pregnancy rates, as well as embryo development. Case The present case report describes the successful pregnancy after testicular sperm aspiration (TESA) combined with intracytoplasmic sperm injection (ICSI) (TESA-ICSI) in a couple where the male presented high sperm DNA fragmentation. In order to sort damaged sperm presenting DNA fragmentation, magnetic activated cell sorting (MACS) with annexin V microbeads (MACS Miltenyi Biotec, Teterow, Germany) was used. Conclusion The authors present the first description of a successful medical case using TESA-ICSI annexin V sperm sorting. Additionally, a follow-up of the child at the age of 4 years old was done.

Healthy Baby Born to a Robertsonian Translocation Carrier following Next-Generation Sequencing-Based Preimplantation Genetic Diagnosis: A Case Report.

Preimplantation genetic diagnosis (PGD) is well established method for treatment of genetic problems associated with infertility. Moreover, PGD with next-generation sequencing (NGS) provide new possibilities for diagnosis and new parameters for evaluation in, for example, aneuploidy screening. The aim of the study was to report the successful pregnancy outcome following PGD with NGS as the method for 24 chromosome aneuploidy screening in the case of Robertsonian translocation. Day 3 embryos screening for chromosomal aneuploidy was performed in two consecutive in vitro fertilization (IVF) cycles, first with fluorescent in situ hybridization (FISH), and then with NGS-based protocol. In each IVF attempt, three embryos were biopsied. Short duration of procedures enabled fresh embryo transfer without the need for vitrification. First IVF cycle with the embryo selected using PGD analysis with the FISH method ended with pregnancy loss in week 8. The second attempt with NGS-based aneuploidy screening led to exclusion of the following two embryos: one embryo with 22 monosomy and one with multiple aneuploidies. The transfer of the only euploid blastocyst resulted in the successful pregnancy outcome. The identification of the euploid embryo based on the NGS application was the first successful clinical application of NGS-based PGD in the case of the Robertsonian translocation carrier couple.

Two new automated, compared with two enzyme-linked immunosorbent, antimüllerian hormone assays.

OBJECTIVE: To compare new automated antimüllerian hormone (AMH) assay performance characteristics from the new automated Elecsys AMH (Roche; Elecsys) and Access AMH (Beckman Coulter; Access) assays with the existing AMH Gen II ELISA (enzyme-linked immunosorbent assay; Gen II; Beckman Coulter) and AMH ELISA (Ansh Labs) assays. DESIGN: Prospective assay evaluation. SETTING: University-affiliated clinical chemistry laboratory. PATIENT(S): Patients referred for serum AMH measurement (n = 83) before start of in vitro fertilization cycle between September 2014 and October 2014. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Serum AMH concentration. RESULT(S): Intra-assay coefficients of variation were low; Ansh ≤ 9.0%; Gen II ≤ 5.8%; Access ≤ 10.7%; and Elecsys ≤ 2.8%. The Passing-Bablok regression equations (pmol/L) were y (Access) = 0.128 + (0.781 × Gen II); and y (Access) = 0.302 + (0.742 x Ansh). For y (Elecys) = 0.087 + (0.729 x Gen II) and y (Elecys) = 0.253 + (0.688 x Ansh Labs). For y (Elecys) = 0.943 - (0.037 × Access). For all the assays, AMH exhibited a moderate positive correlation with AFC (r = 0.62-0.64); number of cumulus oocyte complexes (r = 0.60-0.64); and metaphase II oocytes (r = 0.48-0.50). Accuracy of pregnancy prediction, as determined by area under the receiver operating characteristic curve, was uniformly low for all assays (0.62-0.63). CONCLUSION(S): The novel automated assays exhibit strong concordance in calibration, but derived values are substantially lower than those obtained from pre-existing assays, with assay-specific interpretation required for routine clinical use. These results highlight the need for an international standard of measurement of AMH.

Estradiol Valerate Pretreatment in Short Protocol GnRH-Agonist Cycles versus Combined Pretreatment with Oral Contraceptive Pills in Long Protocol GnRH-Agonist Cycles: A Randomised Controlled Trial.

The strategy of in vitro fertilization (IVF) procedures relies on the increasing pregnancy rate and decreasing the risk of premature ovulation and ovarian hyperstimulation syndrome. They are also designed to avoid weekend oocyte retrievals. Combined oral contraceptive (OC) pills are among the medicines used to accomplish these objectives. Alternatively, estradiol can be used instead of OC to obtain similar results. The aim of our study was to compare the differences in pregnancy rates (PRs), implantation rates, and miscarriage rates between a short agonist protocol with estradiol priming and a long protocol with combined OC. Of the 298 women who participated in this study, 134 achieved clinical pregnancies (45.0%). A higher PR (58.4%, n = 80, compared to 40.3%, n = 54) was achieved in the long protocol after OC pretreatment group. The implantation rate was also higher for this group (37.8% versus 28.0%; P = 0.03). The miscarriage rate was 15.0% (n = 12) for the long protocol after OC pretreatment group and 20.4% (n = 11) for the short agonist group (P = 0.81). The short agonist protocol required a 5.7% lower human menopausal gonadotropin (hMG) dosage than the long protocol but surprisingly the number of oocytes retrieved was also smaller.

Anti-Müllerian hormone (AMH) is a strong predictor of live birth in women undergoing assisted reproductive technology.

In the present study, we evaluated the clinical value of the following parameters: basal anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH), inhibin B and antral follicle count (AFC) in predicting live birth outcomes. The study involved 603 women undergoing in vitro fertilization (IVF) using the long protocol for controlled ovarian hyperstimulation (COH). Serum levels of AMH, FSH and inhibin B as well as AFC were measured on the first three days of the menstrual cycle prior to the beginning of stimulation. AMH was the only independent parameter that correlated with the chance of live birth. We found that live birth rates of 46.2% (patient age <35 years), 44.7% (35-37 years), 32.1% (38-39) and 15.3% (>39) were associated with concentrations of AMH>1.4 ng/ml. For the AMH range 0.6-1.4 ng/ml, the live birth rates were 29.3%, 12.5%, 5.6% and 2.7%, respectively, and for AMH concentrations below 0.6 ng/ml the rates were 7.1%, 8.3%, 0% and 5.8%, respectively. Independently of other parameters affecting the chance of live birth, the success rate was the highest when the AMH level was >2 ng/ml, significantly lower when the AMH concentration was about 1 ng/ml, and 0% when the AMH concentration was ∼0.1 ng/ml. In conclusion, this is the first report to demonstrate that AMH level correlated better than age, FSH or inhibin B concentrations or AFC with live birth outcome. Therefore, the basal serum concentration of AMH may become a new, substantial prognostic factor regarding live birth above and beyond other currently available predictors of IVF outcome.