Evaluation of structural problems in the application of strict criteria for sperm morphology assessment.

BACKGROUND: The WHO manual for basic semen analysis and ISO 23162 describe sperm morphology assessment as a standard part of semen analysis. Older studies showed a correlation between morphology results and (artificial) conception. In more recent studies this relationship was less apparent and there is more emphasis on sperm morphology as a marker for healthy spermatogenesis (and general male health). Meantime, many laboratories ceased morphology assessment, probably due to unfamiliarity with this paradigmatic shift and to technical difficulties in the assessment, like the interpretation of morphological criteria. OBJECTIVES: The aim of this study was to identify morphological criteria with high variability in results in the Dutch External Quality Control (EQC) program. MATERIAL AND METHODS: Over the period 2015-2020, a total of 72 photos of sperm cells along with dichotomous propositions based on 14 criteria as defined in WHO5 (2010) were distributed in the Dutch EQC program for semen analysis. The EQC results were evaluated for variability per criterion and for trends in time. RESULTS: Between 2015 and 2020, 40 to 60 laboratories assessed the photos. Criteria with low variability between participants were related to acrosomal vacuoles, excessive residual cytoplasm, and tail metrics. In contrast, head ovality, regularity of head and midpiece contours, and alignment of the major axis of the midpiece and head led to the highest variability in outcomes. In general, there was a slightly positive trend (lower variability) in time, except for the criteria with the highest variability (stable or declining trend). DISCUSSION AND CONCLUSION: This study indicates that there are (high) variabilities in the interpretation of the morphological criteria, leading to inconsistent outcomes of morphology assessment. The results are discussed from the perspective of imperfections in definitions and examples of the criteria as given in the WHO manuals.

Separation of spermatozoa from erythrocytes using their tumbling mechanism in a pinch flow fractionation device.

Men suffering from azoospermia can father a child, by extracting spermatozoa from a testicular biopsy sample. The main complication in this procedure is the presence of an abundance of erythrocytes. Currently, the isolation of the few spermatozoa from the sample is manually performed due to ineffectiveness of filtering methods, making it time consuming and labor intensive. The spermatozoa are smaller in both width and height than any other cell type found in the sample, with a very small difference compared with the erythrocyte for the smallest, making this not the feature to base the extraction on. However, the length of the spermatozoon is 5× larger than the diameter of an erythrocyte and can be utilized. Here we propose a microfluidic chip, in which the tumbling behavior of spermatozoa in pinched flow fractionation is utilized to separate them from the erythrocytes. We show that we can extract 95% of the spermatozoa from a sample containing 2.5% spermatozoa, while removing around 90% of the erythrocytes. By adjusting the flow rates, we are able to increase the collection efficiency while slightly sacrificing the purity, tuning the solution for the available sample in the clinic.

Influence of embryo culture medium (G5 and HTF) on pregnancy and perinatal outcome after IVF: a multicenter RCT.

STUDY QUESTION: Does embryo culture medium influence pregnancy and perinatal outcome in IVF? SUMMARY ANSWER: Embryo culture media used in IVF affect treatment efficacy and the birthweight of newborns. WHAT IS KNOWN ALREADY: A wide variety of culture media for human preimplantation embryos in IVF/ICSI treatments currently exists. It is unknown which medium is best in terms of clinical outcomes. Furthermore, it has been suggested that the culture medium used for the in vitro culture of embryos affects birthweight, but this has never been demonstrated by large randomized trials. STUDY DESIGN, SIZE, DURATION: We conducted a multicenter, double-blind RCT comparing the use of HTF and G5 embryo culture media in IVF. Between July 2010 and May 2012, 836 couples (419 in the HTF group and 417 in the G5 group) were included. The allocated medium (1:1 allocation) was used in all treatment cycles a couple received within 1 year after randomization, including possible transfers with frozen-thawed embryos. The primary outcome was live birth rate. PARTICIPANTS/MATERIALS, SETTING, METHODS: Couples that were scheduled for an IVF or an ICSI treatment at one of the six participating centers in the Netherlands or their affiliated clinics. MAIN RESULTS AND THE ROLE OF CHANCE: The live birth rate was higher, albeit nonsignificantly, in couples assigned to G5 than in couples assigned to HTF (44.1% (184/417) versus 37.9% (159/419); RR: 1.2; 95% confidence interval (CI): 0.99-1.37; P = 0.08). Number of utilizable embryos per cycle (2.8 ± 2.3 versus 2.3 ± 1.8; P < 0.001), implantation rate after fresh embryo transfer (20.2 versus 15.3%; P < 0.001) and clinical pregnancy rate (47.7 versus 40.1%; RR: 1.2; 95% CI: 1.02-1.39; P = 0.03) were significantly higher for couples assigned to G5 compared with those assigned to HTF. Of the 383 live born children in this trial, birthweight data from 380 children (300 singletons (G5: 163, HTF: 137) and 80 twin children (G5: 38, HTF: 42)) were retrieved. Birthweight was significantly lower in the G5 group compared with the HTF group, with a mean difference of 158 g (95% CI: 42-275 g; P = 0.008). More singletons were born preterm in the G5 group (8.6% (14/163) versus 2.2% (3/137), but singleton birthweight adjusted for gestational age and gender (z-score) was also lower in the G5 than in the HTF group (-0.13 ± 0.08 versus 0.17 ± 0.08; P = 0.008). LIMITATIONS, REASONS FOR CAUTION: This study was powered to detect a 10% difference in live births while a smaller difference could still be clinically relevant. The effect of other culture media on perinatal outcome remains to be determined. WIDER IMPLICATIONS OF THE FINDINGS: Embryo culture media used in IVF affect not only treatment efficacy but also perinatal outcome. This suggests that the millions of human embryos that are cultured in vitro each year are sensitive to their environment. These findings should lead to increased awareness, mechanistic studies and legislative adaptations to protect IVF offspring during the first few days of their existence. STUDY FUNDING/COMPETING INTERESTS: This project was partly funded by The NutsOhra foundation (Grant 1203-061) and March of Dimes (Grant 6-FY13-153). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: NTR1979 (Netherlands Trial Registry). TRIAL REGISTRATION DATE: 1 September 2009. DATE OF FIRST PATIENT'S ENROLMENT: 18 July 2010.

Predictive value of sperm morphology and progressively motile sperm count for pregnancy outcomes in intrauterine insemination.

OBJECTIVE: To investigate the value of sperm parameters to predict an ongoing pregnancy outcome in couples treated with intrauterine insemination (IUI), during a methodologically stable period of time. DESIGN: Retrospective, observational study with logistic regression analyses. SETTING: University hospital. PATIENT(S): A total of 1,166 couples visiting the fertility laboratory for their first IUI episode, including 4,251 IUI cycles. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm morphology, total progressively motile sperm count (TPMSC), and number of inseminated progressively motile spermatozoa (NIPMS); odds ratios (ORs) of the sperm parameters after the first IUI cycle and the first finished IUI episode; discriminatory accuracy of the multivariable model. RESULT(S): None of the sperm parameters was of predictive value for pregnancy after the first IUI cycle. In the first finished IUI episode, a positive relationship was found for ≤4% of morphologically normal spermatozoa (OR 1.39) and a moderate NIPMS (5-10 million; OR 1.73). Low NIPMS showed a negative relation (≤1 million; OR 0.42). The TPMSC had no predictive value. The multivariable model (i.e., sperm morphology, NIPMS, female age, male age, and the number of cycles in the episode) had a moderate discriminatory accuracy (area under the curve 0.73). CONCLUSION(S): Intrauterine insemination is especially relevant for couples with moderate male factor infertility (sperm morphology ≤4%, NIPMS 5-10 million). In the multivariable model, however, the predictive power of these sperm parameters is rather low.

Status of sperm morphology assessment: an evaluation of methodology and clinical value.

OBJECTIVE: To characterize methodological changes in sperm morphology assessment and to correlate sperm morphology with clinical outcome. DESIGN: In this observational study, sperm morphology profiles of patients were analyzed. The percentages of morphologically normal spermatozoa were evaluated with respect to changes in morphology assessment criteria; male aging; and prognostic value for outcomes after in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). SETTING: Diagnostic and clinical laboratories. PATIENT(S): A total of 8,846 men who visited the diagnostic laboratory; 133 samples from a sperm bank; and 3,676 IVF/ICSI couples. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The percentage of morphologically normal spermatozoa in semen samples. The regression of the individual morphologically normal cell profiles. The relation between the percentage of normal forms with pregnancy outcome after IVF/ICSI. RESULT(S): The percentage of morphologically normal spermatozoa showed a decrease from roughly 30%-80% in 1984 to 0%-10% since 2004. With added evidence from sperm bank samples, this decrease was found to be attributable mainly to changes in morphology assessment criteria. Furthermore, an intraindividual aging effect of 0.51% per year was observed. A statistically significant relationship was found between decreases in percentage of normal forms and a lower probability of ongoing pregnancies after IVF, although the area under the curve was only 54%. CONCLUSION(S): Methodological changes had a strong effect on the percentage of morphologically normal spermatozoa over the past few decades. In addition, male aging results in decreasing sperm morphology. The percentage of morphologically normal spermatozoa has no prognostic value for individual IVF/ICSI patients.

Intra- and interobserver analysis in the morphological assessment of early stage embryos during an IVF procedure: a multicentre study.

BACKGROUND: Quality control programs are necessary to maintain good clinical practice. Embryo grading has been described as one of the external quality assurance schemes. Although the evaluation of embryos is based on the assessment of morphological characteristics, considerable intra- and inter-observer variability has been described. In this multicentre study, the variability in the embryo evaluation has been evaluated using morphological characteristics on day 1, day 2 and day 3 of embryo development. METHODS: Five embryologists of four different IVF centers participated in this study. Multilevel images of embryos were presented on a website at different time points to evaluate intra-and inter-observer agreement in the assessment of embryo morphology. The embryos were evaluated on day 1, day 2 and day 3 of their development and each embryologist had to decide if the embryo had to be transferred, cryopreserved or discarded. RESULTS: Both intra-observer agreement and inter-observer agreement were good to excellent for the position of the pronuclei on day 1, the number of blastomeres on day 2 and day 3 and the clinical decision (transfer, cryopreservation, discard). For all other characteristics (size of pronuclei, presence of cytoplasomic halo, degree of fragmentation and size of blastomeres) the intra- and inter-observer agreement was moderate to very poor. CONCLUSIONS: Mono- or multicentre quality control on embryo scoring by morphological assessment can easily be performed through the design of a simple website. In the future the website design can be adapted to generate statistical feedback upon scoring and can even include a training module.

Deciding how many embryos to transfer after in vitro fertilisation: development and pilot test of a decision aid.

OBJECTIVE: When deciding how many embryos to transfer during in vitro fertilisation (IVF), clinicians and patients have to balance optimizing the chance of pregnancy against preventing multiple pregnancies and the associated complications. This paper describes the development and pilot test of a patient decision aid (DA) for this purpose. METHODS: The development of the DA consisted of a literature search, establishment of the format, and a pilot test among IVF patients. The DA development was supervised by a panel of experts in the fields of subfertility, obstetrics and DA-research and it was based on the criteria of the International Patient Decision Aid Standards. RESULTS: One Cochrane review and 34 articles were selected for the DA content. The DA presents information in text, summaries, tables, figures and through an interactive worksheet. The DA was reviewed positively and as acceptable for use in clinical practice by patients and professionals. CONCLUSION: The DA was thoroughly developed and is likely to be helpful for the decision-making process for the number of embryos transferred after IVF. PRACTICE IMPLICATIONS: Physicians and researchers can use the DA without restriction in clinical practice or research related to decision-making.

Review of the role of the plasminogen activator system and vascular endothelial growth factor in subfertility.

OBJECTIVE: To assess the importance of the plasminogen activator (PA) system and vascular endothelial growth factor (VEGF) in subfertility. DESIGN: Review. SETTING: Two university IVF centers. INTERVENTION(S): Systematic literature search (MEDLINE, Science Direct, and bibliographies of published works). RESULT(S): The PA system in the male is involved in the passage of spermatozoa precursor cells over the blood-testis barrier, the epididymal maturation of spermatozoa, the modifications of the sperm surface for capacitation, the acrosome reaction and zona pellucida attachment, and finally the facilitation of spermatozoa to move into the fallopian tubes. In the female, the PA system plays a role during ovulation in the release of the oocyte from the follicle, the facilitation of oocyte movement into the fallopian tubes, and the extracellular matrix degradation important for angiogenesis in the ovary. The function of VEGF during fertilization is largely unknown. It has been suggested that VEGF is important for the fluid and proteins in semen and fluid secretion in the female genital tract, thereby influencing sperm motility and survival. The latter could be due to an effect of VEGF on testicular microvasculature, through which an adequate microenvironment for spermatogenesis is provided. In the female, VEGF is involved in the regulation of the cyclic ovarian angiogenesis, the development and/or selection of follicles, the accumulation of follicular fluid, and corpus luteum angiogenesis. CONCLUSION(S): The role of the PA system and VEGF in reproduction most likely is of great interest. However, much of the data are derived from experimental animal studies. So far, information on the importance of these systems in humans is scarcely investigated. Therefore, further research is required to elucidate the role of the PA system and VEGF in the pathogenesis and prevention of male and female subfertility. Eventually, this will contribute to the improvement of the diagnosis of subfertility and may possibly lead to targeted therapeutic management of subfertility.

Two cycles with single embryo transfer versus one cycle with double embryo transfer: a randomized controlled trial.

BACKGROUND: With the aim of reducing the number of multiple pregnancies after IVF we investigated the effectiveness of two cycles with single embryo transfer (SET) and one cycle with double embryo transfer (DET) after IVF and calculated the cost-effectiveness of both strategies. METHODS: A randomized controlled trial was performed in 107 women, aged <35 years, in their first IVF cycle, with at least one good quality embryo. They were randomized to the SET (n = 54) or DET (n = 53) group using a computer-generated random block number table, stratified for primary or secondary infertility. RESULTS: The cumulative live birth rates per woman randomized of two consecutive cycles of SET [41%; 95% confidence interval (CI) 27-54] versus one cycle of DET (36%; 95% CI 23-49) were comparable, whereas the multiple pregnancy rate was significantly higher: 37% (95% CI 15-59) in the DET and 0% in the in the SET group (P = 0.002). Combining the medical costs of the IVF treatments (where 1.5 more SET cycles were required to achieve each live birth) and of pregnancies up to 6 weeks after delivery, the total medical costs of DET per live birth were 13,680 and 13,438 for SET. CONCLUSIONS: Two cycles with SET were equally effective as one cycle with DET, and the medical costs per live birth up to 6 weeks after delivery were the same. However, if lifetime costs for severe handicaps are included, more than 7000 per live birth will be saved after implementing SET. Because of the high probability of multiple pregnancies in this group of IVF patients, only SET should be performed.

Evaluation of ICSI-selected epididymal sperm samples of obstructive azoospermic males by the CKIA system.

The objective of this study was to evaluate nuclear normality in intracytoplasmic sperm injection (ICSI)-selected epididymal sperm from obstructive azoospermic (OA) patients. We evaluated whether the selection criteria used in routine ICSI (morphology and motility at a magnification of 400x) is adequate for selecting "normal" sperm from epididymal samples. Surgically retrieved spermatozoa from the caput epididymis of 15 OA patients and ejaculated sperm samples from 9 normospermic donors were evaluated with a DNA-specific stain (Feulgen) and in combination with the computerized karyometric image analysis (CKIA) system. Original (unselected) samples and ICSI-selected sperm were compared in donor and patient samples. In the original fraction, a larger variation in almost all measured parameters was found in epididymal sperm than in ejaculated sperm. After sperm selection, the morphometry was comparable between epididymal and ejaculated sperm. However, for those parameters related to the DNA stainability and chromatin texture (nuclear condensation), significant differences between patients and donors were observed. This result suggests that the size and form of the sperm do not necessarily hold similar internal structures. Thus, the frequency of "normal" sperm significantly increased after ICSI selection, but the improvement was more marked in donor than in OA patients' samples. In conclusion, at least a twofold increase in the number of normal spermatozoa was achieved after ICSI selection. The heterogeneity in the stainability and chromatin condensation of epididymal samples from OA patients indicates that some of the selected spermatozoa have a hypocondensed or hypercondensed chromatin. Even in the best of donor cases, no more than 55% of the selected sperm scored normal with CKIA, indicating that the present routine ICSI selection criteria are not sufficient for selecting normal condensed nuclei.

Fertility in men with testicular germ cell tumors.

OBJECTIVE: To assess the prevalence of fertility or infertility in men before and after treatment for unilateral testicular cancer. The results were compared with the lifetime prevalence of infertility in the general population (20%-28%). DESIGN: Survey. SETTINGS: University referral center for testicular cancer. PATIENT(S): Two hundred twenty-six patients treated for testicular cancer. INTERVENTION(S): Questionnaire on fertility and fertility-related factors before and after treatment of disease. MAIN OUTCOME MEASURE(S): Prevalence of fertility before and after treatment for testicular cancer. RESULT(S): Before the cancer was diagnosed, 79 (66%) of 120 couples who attempted to conceive succeeded within 1 year. After treatment, 38 (43%) of 88 couples conceived within 1 year. Seven couples used cryopreserved sperm to conceive a child after treatment. The different treatment modalities did not significantly influence the outcome of patients' wish for children. Congenital malformations were recorded in approximately 4% of the children born before or after treatment. CONCLUSION(S): Although the majority of the patients with testicular cancer have a fulfilled wish with regard to children, it seems to be more difficult to father a child after treatment compared with the case in the general population. Because it is not possible to predict which patient will have fertility problems after treatment, cryopreservation should be offered to every testicular cancer patient. An increased risk for congenital malformations was not observed.

Use of computerized karyometric image analysis for evaluation of human spermatozoa.

The objective of this study was to evaluate a computer image system for its ability to determine morphological and nuclear semen characteristics in an integral and reproducible way. Semen samples from 19 normospermic fertile donors were used to estimate preliminary cutoff values for spermatozoa and to test the reproducibility of the system. Ten aliquots of 1 sample were used to investigate the sensitivity of the system for experimental conditions by exposure to different laboratory variables. Human spermatozoa were stained with Feulgen dye and analyzed with a magnification of 1000x. A panel of 21 parameters was measured for each sperm nucleus using the computerized karyometric image analysis (CKIA) system. Eight parameters were found to be sensitive for differentiating normal or abnormal human spermatozoa, and cutoff values for each parameter were defined for quantitative analysis. These 8 parameters were grouped into 3 categories depending on their descriptive value: morphometry, DNA condensation (stainability), and chromatin texture. Intrapatient and interpatient variabilities were tested by calculating the reliability coefficient for each of the 8 parameters as well as for each category. Reliability coefficients were all >70% (indicative of the suitability of the system to identify differences between spermatozoa). Interpatient variability (SD) was 5%. Although it was not statistically significant, a variation of 10.9% in measurements was found when the effects of experimental conditions were tested. We conclude that an objective description of the human sperm nucleus can be achieved with CKIA, yielding high interpatient and intrapatient reliability coefficients (reproducibility), thereby adding a new tool for the quantification of normal sperm.