Comment on 'Importance of real-time measurement of sperm head morphology in intracytoplasmic sperm injection' by Fumiaki Itoi et al.

We present a commentary on the article published in the Zygote FirstView: 'Importance of real-time measurement of sperm head morphology in intracytoplasmic sperm injection' by Fumiaki Itoi and colleagues. We comment on the importance of providing the microscope setup details whenever sperm morphology visualization is discussed. The claim of ×6000-10,000 magnification is misleading as such levels of magnification are impossible to achieve.

Mitochondrial DNA Copy Number in Cleavage Stage Human Embryos-Impact on Infertility Outcome.

A retrospective case control study was undertaken at the molecular biology department of a private center for reproductive medicine in order to determine whether any correlation exists between mitochondrial DNA (mtDNA) content of cleavage-stage preimplantation embryos and their developmental potential. A total of 69 couples underwent IVF treatment (averaged women age: 36.5, SD 4.9) and produced a total of 314 embryos. A single blastomere was biopsied from each embryo at the cleavage stage (day-3 post-fertilization) subjected to low-pass next generation sequencing (NGS), for the purpose of detecting aneuploidy. For each sample, the number of mtDNA reads obtained after analysis using NGS was divided by the number of reads attributable to the nuclear genome. The mtDNA copy number amount was found to be higher in aneuploid embryos than in those that were euploid (mean mtDNA ratio ± SD: 6.3 ± 7.5 versus 7.1 ± 5.8, p < 0.004; U Mann−Whitney test), whereas no statistically significant differences in mtDNA content were seen in relation to embryo morphology (6.6 ± 4.8 vs. 8.5 ± 13.6, p 0.09), sex (6.6 ± 4.1 vs. 6.2 ± 6.8, p 0.16), maternal age (6.9 ± 7.8 vs. 6.7 ± 4.5, p 0.14) or its ability to implant (7.4 ± 6.6 vs. 5.1 ± 4.6, p 0.18). The mtDNA content cannot serve as a useful biomarker at this point in development. However, further studies investigating both quantitative and qualitative aspects of mtDNA are still required to fully evaluate the relationship between mitochondrial DNA and human reproduction.

Role of Leptin and Adiponectin in Endometrial Cancer.

Endometrial cancer is the most common malignancy of the female genital tract. Obesity is a strong risk factor for endometrial cancer. Adipose tissue is an active endocrine organ that synthesizes biologically active cytokine peptides, called adipokines. Adiponectin and leptin are the main cytokines of adipose tissue, which may influence the development of metabolic diseases and carcinogenesis. In this scenario, we describe the role of leptin and adiponectin in the development of endometrial cancer. A better understanding of the signalling pathway of these cytokines in endometrial cancerogenesis will provide an opportunity for effective target therapy and may be usable in fertility-sparing treatment. In the future, clinical trials focusing on adipokines, molecular biology, and genetics of the tumour will be needed.

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.

Age-related decline in AMH is assay dependent limiting clinical interpretation of repeat AMH measures across the reproductive lifespan.

PURPOSE: The aim of the study was to determine whether the assays exhibit an interaction with age and exhibit heterogeneous age related declines in AMH. Apart of chronological age, AMH variation was investigated with relation to menstrual cycle day (MCD). The goal implicates two questions: Are distributions of AMH concentrations homogenous after adjustment for the specific AMH assay? Does age-assay product has an effect on AMH depletion? METHODS: The study was conducted by examining results of AMH tests performed for 12,917 women with four types of AMH assays: Immunotech I generation kit (IMI, 4016 samples), Beckman Coulter II generation kit RUO (BCII RUO, 3430 samples), Beckman Coulter II generation kit with IVD certificate (BCII IVD, 830 samples), and Ansh Labs I generation kit (AnshLabs, 4641 samples). Statistical analysis included ACNOVA and least square regression technique. RESULTS: Menstrual cycle day has no effect on AMH measurements. On the other hand, AMH values differed substantially between the four assays, with a marked discordance in the rate of age-related AMH decline for the four assays (ranging from -8.16% (95% CI: -8.79, -7.54) to -11.53% (95% CI -12.20, -10.87), with a significant interaction between age and assay. CONCLUSIONS: (1) The distribution of AMH concentration is heterogeneous after controlling the age across assays; (2) the rate of AMH decline as a function of age is different for the four manual AMH ELISA assays.

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.

Current methods for preimplantation genetic diagnosis.

Preimplantation Genetic Diagnosis (PGD) used in assisted reproduction techniques is designed to provide help for couples trying to conceive a child, as it helps deliver healthy offspring. After in vitro fertilization, material is collected from the oocyte (polar body), 3-day-old embryo, or increasingly often, from the trophectoderm of a blastocyst. Selection of the diagnostic method depends on the testing center, but methods such as aCGH (Comparative Genomic Hybridization Array) and NGS (Next-Generation Sequencing) are supposed to have the highest reliability and precision. This paper presents a review of the most important methods used in PGD, their advantages and disadvantages as well as efficacy in the procedures in which they are used.

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.

Humoral Response after Vaccination with Half-Dose of BNT162b2 in Subjects under 55 Years of Age.

In the context of the ongoing COVID-19 pandemic, using a half-dose schedule vaccination can help to return to normalcy in a cost-efficient manner, which is especially important for low and middle-income countries. We undertook a study to confirm that in adults up to 55 years old, the humoral response to the half-dose (15 µg, 35 participants between 18 and 55 years old) and to the recommended dose (30 µg, 155 participants) in the two-dose three-week interval schedule would be comparable. Antibody levels were measured by the Elecsys Anti-SARS-CoV-2 S assay (Roche Diagnostics, upper detection limit: 2570 BAU/mL) on the day of dose 2 of the vaccine and then 8-10 days later to assess peak response to dose 2. The difference in proportions between the participants who did and did not exceed the upper detection limit 8-10 days after dose 2 was not statistically significant (p = 0.152). We suggest that a half-dose schedule can help to achieve widespread vaccination coverage more quickly and cheaply.

Usefulness of IVD Kits for the Assessment of SARS-CoV-2 Antibodies to Evaluate the Humoral Response to Vaccination.

BACKGROUND: The introduction of the vaccination against SARS-CoV-2 infection creates the need for precise tools for the quality control of vaccination procedures, detection of poor humoral response, and estimation of the achieved protection against the disease. Thus, the study aimed to compare the results of the anti-SARS-CoV-2 tests to evaluate the application of the WHO standard unitage (the binding antibody units; BAU/mL) for a measurement of response to the vaccination. METHODS: Patients undergoing vaccination against SARS-CoV-2 with Pfizer/BioNTech BNT162b2 (BNT162b2) (n = 79), referred for SARS-CoV-2 antibody measurement prior to vaccination and 21 days after dose 1, and 8, 14, and 30 days after dose 2 were included. The sera were tested with three assays: Elecsys SARS-CoV-2 S (Roche), LIAISON® SARS-CoV-2 TrimericS IgG (DiaSorin), and SARS-CoV-2 IgG II Quant (Abbott). RESULTS: The three assays showed varying correlations at different time points in the study. The overall agreement for all samples was moderate to high (ρ = 0.663-0.902). We observed the most uniform agreement for the day of dose 2 (ρ = 0.775-0.825), while it was least consistent for day 8 (ρ = -0.131-0.693) and 14 (ρ = -0.247-0.603) after dose 2. The dynamics of changes of the SARS-CoV-2 antibody levels in patients without history of prior SARS-CoV-2 infection appears homogenous based on the Roche results, more heterogenous when considering the DiaSorin results, and in between for the Abbott results. CONCLUSIONS: The results highlight the need for further work on the international standard of measurement of SARS-CoV-2 Ig, especially in the era of vaccination. The serological assays can be useful to detect IgG/IgM antibodies to assess the response to the vaccination. However, they cannot be used interchangeably. In terms of the evaluation of the immune response to the BNT162b2 vaccine, Roche and Abbott kits appear to be more useful.

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.

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.

Decreasing quality of the new generations of anti-Müllerian hormone assays.

Anti-Müllerian hormone (AMH) measurements are widely used to optimize the stimulation protocols. First generation AMH kits correlated well with ovarian reserve and response to stimulation. In the present study we aimed to asses if the new generation kits share the same accurate correlations. Retrospective data were collected from 8323 blood samples. For comparison we used Immunotech I generation kit (ImI 4035 samples), Beckman Coulter II generation kit RUO (BCII RUO 3449, samples) and Beckman Coulter II generation kit with IVD certificate (BCII IVD 839 samples). We compared average AMH concentrations measured with different kits, as well as correlation between kits. We also compared average AMH concentrations in sera collected on different cycle days and samples of different quality of preservation. AMH serum concentrations differed for each kit, ranging 4.4 ± 4.12 (mean ± SD) for the ImI, 2.68 ± 3.15 for the BCII RUO, and 1.64 ± 2.85 for BCII IVD. The mean differences from an adjusted regression model were -48.7%, -40%, and -69.2%, respectively. In conclusion, the changes of the BC AMH kits are unpredictable; however, the improvement of them is still possible. It would be very dangerous to use elaborated stimulation protocol (based on the Ist generation AMH results) with the results from the IInd generation assays.