Is paternal age associated with transfer day, developmental stage, morphology, and initial hCG-rise of the competent blastocyst leading to live birth? A multicenter cohort study.

In this study we investigated whether age of men undergoing assisted reproductive technology (ART) treatment was associated with day of transfer, stage, morphology, and initial hCG-rise of the competent blastocyst leading to a live birth? The design was a multicenter historical cohort study based on exposure (age) and outcome data (blastocyst stage and morphology and initial hCG-rise) from men whose partner underwent single blastocyst transfer resulting in singleton pregnancy/birth. The ART treatments were carried out at sixteen private and university-based public fertility clinics. We included 7246 men and women, who between 2014 and 2018 underwent controlled ovarian stimulation (COS) or Frozen-thawed Embryo Transfer (FET) with a single blastocyst transfer resulting in singleton pregnancy were identified. 4842 men with a partner giving birth were included, by linking data to the Danish Medical Birth Registry. We showed that the adjusted association between paternal age and transfer day in COS treatments was OR 1.06, 95% CI (1.00;1.13). Meaning that for every increase of one year, men had a 6% increased probability that the competent blastocyst was transferred on day 6 compared to day 5. Further we showed that the mean difference in hCG values when comparing paternal age group 30-34, 35-39 and 40-45 with the age group 25-29 in those receiving COS treatment, all showed significantly lower adjusted values for older men. In conclusion we hypothesize that the later transfer (day 6) in female partners of older men may be due to longer time spent by the oocyte to repair fragmented DNA of the sperm cells, which should be a focus of future research in men.

Association between women's age and stage, morphology, and implantation of the competent blastocyst: a multicenter cohort study.

OBJECTIVE: To study if the age of women undergoing assisted reproductive technology treatment associates with stage, morphology, and implantation of the competent blastocyst. DESIGN: Multicenter historical cohort study based on exposure (age) and outcome data (blastocyst stage and morphology and initial human chorionic gonadotrophin [hCG] rise) from women undergoing single blastocyst transfer resulting in singleton pregnancy/birth. SETTING: Sixteen private and university-based facilities. PATIENT(S): In this study, 7,246 women who, between 2014 and 2018, underwent controlled ovarian stimulation (COS) or frozen-thawed embryo transfer (FET) with a single blastocyst transfer resulting in singleton pregnancy were identified. Linking data to the Danish Medical Birth Registry resulted in a total of 4,842 women with a live birth being included. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The competent blastocyst development stage (1-6), inner cell mass (A, B, C), trophectoderm (A, B, C), and initial serum hCG value. RESULT(S): Adjusted analysis of age and stage in COS treatments showed that for every 1-year increase in age there was a 5% reduced probability of the competent blastocyst assessed as being in a high stage at transfer. Comparison between hCG values in women 18-24 years and 25-29 years in both COS and FET showed significantly lower levels in the youngest women. CONCLUSION(S): The initial hCG rise was influenced by the age of the woman, with an identical pattern for hCG values in COS and FET treatments. In COS, the competent blastocyst had a reduced stage with increasing women's age.

[Preimplantation genetic testing for aneuploidy].

This review summarises the current knowledge on preimplantation genetic testing for aneuploidy (PGT-A). Selection and transfer of euploid embryos aim to improve live birth rate (LBR) per embryo transfer, but fluorescence in situ hybridisation-based PGT-A and biopsy of cleavage stage embryos in the 2000s was a disappointment, as studies revealed a reduced LBR. Today, PGT-A includes comprehensive chromosome screening primarily of blastocyst biopsies. The benefit of PGT-A is highly debated: some suggest improved treatment outcome, while others claim, that the procedure is not cost-effective.

[Preimplantation genetic diagnosis].

In Denmark, preimplantation genetic diagnosis (PGD) is offered within the public healthcare to families with a known risk of an inherited disease in a child - as an alternative to prenatal diagnosis. It is a well-established technique with rather well-described perinatal- and neonatal outcomes, being comparable to what is seen following in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). The most common strategy is now to perform trophectoderm biopsy and then vitrify, while the diagnostic test is performed. Until 2013, 134 children have been born following PGD. Today, the clinical pregnancy rates are comparable to those following IVF/ICSI.

A randomized clinical trial to evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium for in vitro fertilization.

OBJECTIVE: To evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium on ongoing implantation rate (OIR). DESIGN: Multicenter, randomized, placebo-controlled, double-blinded prospective design. SETTING: Fourteen Scandinavian fertility clinics. PATIENT(S): A total of 1,332 women with indication for in vitro fertilization or intracytoplasmic sperm injection; 1,149 received embryo transfer (GM-CSF: n = 564; control: n = 585). INTERVENTION(S): Oocytes were fertilized, and embryos cultured and transferred in control medium or test medium containing 2 ng/mL GM-CSF. MAIN OUTCOME MEASURE(S): OIR at gestational week 7, with follow-up at week 12 and birth. RESULT(S): At week 7, OIRs were 23.5% (GM-CSF), and 20.0% (control) (odds ratio [OR] 1.26, 95% confidence interval [CI] 0.91-1.75). At week 12, OIRs were 23.0% (GM-CSF) and 18.7% (control) (OR 1.35, 95% CI 1.06-1.72), and live birth rates were 28.9% and 24.1%, respectively (OR 1.35, 95% CI 1.03-1.78). The effect of GM-CSF was influenced by the human serum albumin concentration in the medium. Birth weight and abnormality incidence were similar in both groups. Exploratory analyses showed that GM-CSF increased OIR in women with previous miscarriage, especially in women with more than one miscarriage. CONCLUSION(S): Addition of GM-CSF to embryo culture medium elicits a significant increase in survival of transferred embryos to week 12 and live birth. Our results are consistent with a protective effect of GM-CSF on culture-induced embryo stress. GM-CSF may be particularly efficacious in women with previous miscarriage. CLINICAL TRIAL REGISTRATION NUMBER: NCT00565747.

Traditional detection versus computer-controlled multilevel analysis of nuclear structures from donated human embryos.

BACKGROUND: Multinuclearity is known to correlate with decreased implantation and pregnancy rates. Thus, a valid detection of nuclear structures especially among otherwise good quality embryos may be of great importance in order to improve clinical outcome. In this study, we have compared traditional manual microscopic analysis with computer-controlled multilevel morphological assessment for analysis of nuclear status in human embryos. METHODS: In total, 84 donated 2- and 4-cell embryos with < or = 20% fragmentation from patients referred for IVF or ICSI treatment were included. Mono- and multinuclearity was recorded using traditional analysis as well as computer-controlled multilevel analysis of each intact embryo. Subsequently, the embryos were separated into individual blastomeres to assess the number of nuclear structures. All nuclear structures were fixed and stained for DNA. RESULTS: There was no significant difference (P = 1.0) between embryonic nuclear status detected by computer-controlled analysis of the intact embryos and of the separated blastomeres. Additionally, 100% of the fixed nuclear structures contained DNA. However, using traditional morphological analysis, significantly more embryos (26%) had incorrect nuclear status detected (P = 0.002). Further, the presence of <10% embryonic fragmentation had no impact on the correct detection of nuclear structures using the multilevel analysis. For embryos with 11-20% fragmentation, 86% of the nuclear structures detected in the separated blastomeres were found in the intact embryos. The mean diameter of nuclear structures was significantly decreased from 22.1 microm in mononucleate 2-cell embryos to 18.7 microm in mononucleate 4-cell embryos (P < 0.001). CONCLUSION: The results of this study indicate that the use of computer-controlled multilevel morphological analysis can improve the detection of nuclear structures in human embryos.

Total cytoplasmic volume as biomarker of fragmentation in human embryos.

PURPOSE: To use computer-controlled, multilevel embryo morphology analysis to quantify the reduction in cytoplasmic volume from the zygote stage to the combined volume of the individual blastomeres as an expression for the degree of fragmentation. METHODS: Zygotes and their corresponding embryos from patients referred to ICSI treatment were analyzed. Sequences of digital images were taken focussing at 5-microm intervals through the zygotes and embryos. Assessment of cytoplasmic volumes and fragmentation were based on these sequences. RESULTS: The mean cytoplasmic reduction of the embryos were significant linear increasing with increasing degree of fragmentation assessed by traditional evaluation (P < 0.001). Embryos scored to be highly fragmented had on average a cytoplasmic reduction of 63% larger than embryos assessed to have no fragmentation. In total, 68% of the embryos had a cytoplasmic reduction lying within their allocated fragmentation group. CONCLUSION: Assessment of embryonic fragmentation based on computer-controlled, multi-level embryo analysis of the blastomere volumes expressed in relation to the volume of the preceding zygote may allow a more precise and standardized evaluation of fragmentation than the traditional fragmentation assessment.

Computer-controlled, multilevel, morphometric analysis of blastomere size as biomarker of fragmentation and multinuclearity in human embryos.

BACKGROUND: Little is known about blastomere size at different cleavage stages and its correlation with embryo quality in human embryos. Using a computer system for multilevel embryo morphology analysis we have analysed blastomeres of human embryos and correlated mean blastomere size with embryonic fragmentation and multinuclearity. METHODS: A consecutive cohort of 232 human 2-, 3- and 4-cell embryos from patients referred for ICSI treatment were included. Sequences of digital images were taken by focusing at 5- micro m intervals through the embryo. Blastomere sizes and number of nuclear structures were evaluated based on these sequences. The degree of embryonic fragmentation was evaluated by normal morphological assessment prior to transfer and correlated to the blastomere sizes. RESULTS: As a result of normal cell cleavage, mean blastomere size decreased significantly from a volume of 0.28 x 10(6) microm(3) at the 2-cell stage to 0.15 x 10(6) microm(3) at the 4-cell stage (P < 0.001). Mean blastomere size decreased significantly (P < 0.001) with increasing degree of embryonic fragmentation, where highly fragmented embryos showed a 43-67% reduction in blastomere volume compared with embryos with no fragmentation. Multinucleated blastomeres were significantly larger than non-multinucleated blastomeres (P < 0.001). On average, multinucleated blastomeres were 51.5, 67.8 and 73.1% larger than their non-multinucleated sibling blastomeres at the 2-, 3- and 4-cell stage, respectively. Furthermore, the average volume of non-multinucleated blastomeres originating from multinucleated embryos was significantly smaller than the average volume of the blastomeres from mononucleated embryos (P < 0.001). CONCLUSIONS: The results of this study show that the average blastomere size is significantly affected by degree of fragmentation and multinuclearity, and that computer-assisted, multilevel analysis of blastomere size may function as a biomarker for embryo quality.