The preparation and culture of washed human sperm: a comparison of a suite of protein-free media with media containing human serum albumin

Objective To compare two suites of culture media (one with HSA and one protein-free (PF) supplemented with methylcellulose) for washing human sperm in IVF. Methods Semen samples (n = 41) underwent parallel density gradient preparation using PF or HSA-supplemented culture medium and subsequent yield, survival, morphology and motility were compared. Results The PF medium resulted in a significantly higher sperm yield (P \u3c 0.0001), but similar sperm morphology (P = 0.822) and 24-h survival (P = 0.11). There was, however, a lower percentage of progressively motile sperm (P \u3c 0.0001) and a higher proportion of sperm demonstrating non-progressive motility (P \u3c 0.0001) in the PF medium when observed on a Makler Chamber, apparently an artefact as a similar sperm motility index was measured using a Sperm Quality Analyser (P = 0.83). Attachment of sperm in PF medium to the glass chamber reduced with time and any differences had disappeared after 6 min on the counting chamber. Conclusion These results support the use of PF media supplemented with methylcellulose as an alternative to HSA, although a modification to the manufacturer\u27s protocol of 6-min pre-incubation before assessing sperm motility must be used. Further studies should investigate the function of such sperm prepared in PF medium

Spontaneous collapse as a prognostic marker for human blastocysts: A systematic review and meta-analysis

STUDY QUESTION: Is spontaneous collapse (SC) by human blastocysts a prognostic factor in IVF treatment? SUMMARY ANSWER: SC in human blastocyst is associated with reduced euploid embryo and pregnancy rates. WHAT IS KNOWN ALREADY: SC of the human blastocyst is a phenomenon that was revealed relatively recently following the clinical application of time-lapse monitoring in IVF laboratories. The ploidy and clinical prognosis of affected blastocysts are still poorly understood, with inconsistent reports. Systematic reviews and meta-analyses on this topic are currently absent in the literature but its potential as a marker of embryo viability holds great clinical value. In this study, we aimed to comprehensively evaluate the potential of SC as a prognostic factor in regard to ploidy status, and pregnancy, live birth and miscarriage rates. STUDY DESIGN, SIZE, DURATION: A systematic review and meta-analysis were performed according to PRISMA guidelines, with a protocol registered with PROSPERO (CRD42022373749). A search of MEDLINE, EMBASE, and the Cochrane Library for relevant studies was carried out on 10 October 2022, using key words relevant to \u27blastocyst collapse\u27 and \u27time-lapse imaging\u27. PARTICIPANTS/MATERIALS, SETTING, METHODS: Two independent reviewers systematically screened and evaluated each study in terms of participants, exposure, comparator, and outcomes (PECO). The Quality In Prognosis Studies tool was used for quality assessment. Data were extracted according to Cochrane methods. Pregnancy, live birth, ploidy, or miscarriage data were summarized by risk ratios (RRs) or odds ratios and their 95% CIs. All meta-analyses were performed with random-effects models. MAIN RESULTS AND THE ROLE OF CHANCE: Following removal of duplicates, a total of 196 records were identified by the initial search. After screening according to PECO, 19 articles were included for further eligibility assessment. For meta-analysis, seven retrospective cohort studies were eventually included. After data pooling, the incidence of blastocyst SC was 37.0% (2516/6801) among seven studies (ranging from 17.4% to 56.2%). SC was associated with significantly lower clinical pregnancy rates (two studies, n = 736; RR = 0.77, 95% CI = 0.62-0.95; I 2 = 30%), ongoing pregnancy rates (five studies, n = 2503; RR = 0.66, 95% CI = 0.53-0.83; I 2 = 60%), and reduced euploidy rates (three studies, n = 3569; RR = 0.70, 95% CI = 0.59-0.83; I 2 = 69%). Nevertheless, live birth rates (two studies, n = 816; RR = 0.76, 95% CI = 0.55-1.04; I 2 = 56%) and miscarriage rate (four studies, n = 1358; RR = 1.31, 95% CI = 0.95-1.80; I 2 = 0%) did not differ between blastocysts with or without SC. There was, however, significant heterogeneity between the studies included for evaluation of ongoing pregnancy rates (I 2 = 60%, P = 0.04), live birth rates (I 2 = 56%, P = 0.13), and ploidy rates (I 2 = 69%, P = 0.04). Subgroup analyses were conducted according to different definitions of SC, number of collapse events, and whether the transferred blastocyst had undergone preimplantation genetic testing for aneuploidy; with inconclusive findings across subgroups. LIMITATIONS, REASONS FOR CAUTION: All studies in the meta-analysis were retrospective with varying levels of heterogeneity for different outcomes. Not all studies had accounted for potential confounding factors, therefore only unadjusted data could be used in the main meta-analysis. Studies employed slightly different strategies when defining blastocyst SC. Standardization in the definition for SC is needed to improve comparability between future studies. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that blastocyst SC has negative implications for a pregnancy. Such blastocysts should be given a low ranking when selecting from a cohort for intrauterine transfer. Blastocyst SC should be considered as a contributing variable when building blastocyst algorithms to predict pregnancy or live birth

Blastocyst transfer for multiple prior IVF failure: a five year descriptive study

Patients with recurrent IVF failure are generally regarded as having a poor prognosis, and when female age exceeds 35yrs such patients face a particularly bleak outlook. This study reported on blastocyst transfer (BT) performed over a five-year interval in patients seeking “second opinion” after multiple failed IVF cycles. Clinical features and reproductive outcomes were compared between two sets of poor-prognosis IVF patients undergoing BT for the first time, the initial group underwent treatment in 2002 (n=66) and a second group presented five years later (n=392). The two clinical sets had no patients in common. The 2002 group had an average of 3.5(±1.1) prior failed IVF cycles at baseline, and mean (±SD) patient age was 36.4(±3.9)yrs. Average number of oocytes retrieved in this group was 10.4(±5.3) with a fertilisation rate of 58.8%. Although embryo arrest resulted in no transfer for 19 patients (28.8%), clinical pregnancy was achieved for 59.6% of transfers. Five years later, 392 patients underwent BT, but this group had an average of 4.5(±2.3) prior failed IVF cycles. Mean (±SD) female age was 36.0(±3.9)yrs, and the average number of oocytes retrieved in this group was 9.1(±5.4); the fertilisation rate was 59.5%. No blastocysts were available for transfer in 99 cases (25.3%); clinical pregnancy was achieved for 50.0% of transfers. The number of blastocysts transferred was similar in the two groups (1.6 vs. 1.3; p=0.06); the twinning rate rose slightly from 8.2% to 15.1% (p=0.12) despite an increased utilisation of single embryo transfer in 2007 (19.7% vs. 22.2%; p=0.40). Comparisons from 2002 and 2007 found no important differences between the two patient groups, except for a significantly higher rate of prior failed cycles in the 2007 group (

The Influence of Sample Volume Applied to the Makler Sperm Counting Chamber Upon the Measured Concentration of Latex Beads: A Multi-Centre Study

Objective: To undertake a multi-centre study to maximize the number of Makler chambers used. Methods: A total of 15 laboratories participated with 31 Makler chambers. A suspension of latex beads was prepared to a concentration of 20 millions per milliliter, and 0.5 mL aliquots distributed to each participating laboratory. They measured the concentration on their Makler chamber(s) used for routine semen analysis by adding 3, 4, 5, 7 and 10 μL volumes of bead suspension to the chamber. Results: There was no difference in within-chamber analysis of the bead concentration according to the volume of bead suspension applied within the range of 3–10 μL (F4,14=2.634, P=0.056). However, the between-chamber effects were significantly different (F30,124=4.937, P=0.000), and 24/31 (77.5%) chambers tested had an average bias\u3e10% compared to the target bead concentration. Conclusions: A volume of 3–10 μL added to Makler counting chambers does not influence the concentration measured of latex beads, but the between-chamber variability and positive bias seen would suggest that other sources of error are present which are yet to be identified

The preparation and culture of washed human sperm: A comparison of a suite of protein-free media with media containing human serum albumin

Objective: To compare two suites of culture media (one with HSA and one protein-free (PF) supplemented with methylcellulose) for washing human sperm in IVF. Methods: Semen samples (n = 41) underwent parallel density gradient preparation using PF or HSA-supplemented culture medium and subsequent yield, survival, morphology and motility were compared. Results: The PF medium resulted in a significantly higher sperm yield (P < 0.0001), but similar sperm morphology (P = 0.822) and 24-h survival (P = 0.11). There was, however, a lower percentage of progressively motile sperm (P < 0.0001) and a higher proportion of sperm demonstrating non-progressive motility (P < 0.0001) in the PF medium when observed on a Makler Chamber, apparently an artefact as a similar sperm motility index was measured using a Sperm Quality Analyser (P = 0.83). Attachment of sperm in PF medium to the glass chamber reduced with time and any differences had disappeared after 6 min on the counting chamber. Conclusion: These results support the use of PF media supplemented with methylcellulose as an alternative to HSA, although a modification to the manufacturer's protocol of 6-min pre-incubation before assessing sperm motility must be used. Further studies should investigate the function of such sperm prepared in PF medium

A survey of scientists’ awareness of and attitudes to the use of human blood products and alternatives in human assisted reproductive technology

<p>Scientists working in assisted reproduction [members of Scientists in Reproductive Technology (SIRT) Australia, and subscribers of the online forums EmbryoMail and Quartec] were invited to complete an online questionnaire on the use of human blood products in assisted reproductive technologies (ART). A total of 260 started the questionnaire, with 208 (80%) completing it. A total of 62% of respondents had worked in human ART ≥8 years and 68% had post-graduate qualifications. The majority (82%) reported using products of animal or human origin, with 75% knowing why protein was added to culture media and 41% not worried by this. Almost half (49%) of respondents were unaware of regulations surrounding the use of human blood products in health care and 70% were unaware of adverse events involving human blood products in human ART. Most respondents (70%) indicated that they were not concerned about infections such as hepatitis, but agents such as prions were a cause for concern (57%). A total of 57% of respondents were unaware of alternatives, but 77% would use a suitable alternative. Using blood products in human ART is surrounded by a lack of awareness, often independent of respondents’ qualifications or experience. A better understanding of these products and possible alternatives is required if informed decisions about their suitability are to be made.</p