Optimized sperm selection: a highly efficient device for the isolation of progressive motile sperm with low DNA fragmentation index.

PURPOSE: To identify the sperm preparation procedure that selects the best sperm population for medically assisted reproduction. METHODS: Prospective observational study comparing the effect of four different sperm selection procedures on various semen parameters. Unused raw semen after routine diagnostic analysis was split in four fractions and processed by four different methods: (1) density gradient centrifugation (DGC), (2) sperm wash (SW), (3) DGC followed by magnetic activated cell sorting (MACS), and (4) using a sperm separation device (SSD). Each fraction was analyzed for progressive motility, morphology, acrosome index (AI), and DNA fragmentation index (DFI). RESULTS: With DGC as standard of care in intraclass correlation coefficient analysis, only SSD was in strong disagreement regarding progressive motility and DFI [0.26, 95%CI (- 0.2, 0.58), and 0.17, 95%CI (- 0.19, 0.45), respectively]. When controlling for abstinence duration, DFI was significantly lower after both MACS and SSD compared to DGC [- 0.27%, 95%CI (- 0.47, - 0.06), p = 0.01, and - 0.6%, 95%CI (- 0.80, - 0.41), p < 0.001, respectively]. Further comparisons between SSD and MACS indicate significantly less apoptotic cells [Median (IQR) 4 (5), 95%CI (4.1, - 6.8) vs Median (IQR) 5 (8), 95%CI (4.9, - 9.2), p < 0.001, respectively] and dead cells [Median (IQR) 9.5 (23.3), 95%CI (13.2, - 22.4) vs Median (IQR) 22 (28), 95%CI (23.1, - 36.8), p < 0.001, respectively] in the SSD group. CONCLUSION: The selection of a population of highly motile spermatozoa with less damaged DNA from unprocessed semen is ideally performed with SSD. Question remains whether this method improves the embryological outcomes in the IVF laboratory.

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation.

Mature oocyte vitrification is the standard of care to preserve fertility in women at risk of infertility. However, ovarian tissue cryopreservation (OTC) is still the only option to preserve fertility in women who need to start gonadotoxic treatment urgently or in prepubertal children. During ovarian cortex preparation for cryopreservation, medullar tissue is removed. Growing antral follicles reside at the border of the cortex-medullar interface of the ovary and are broken during this process, releasing their cumulus-oocyte complex (COC). By thoroughly inspecting the medium and fragmented medullar tissue, these immature cumulus-oocyte complexes can be identified without interfering with the OTC procedure. The ovarian tissue-derived immature oocytes can be successfully matured in vitro, creating an additional source of gametes for fertility preservation. If OTC is performed within or near a medical assisted reproduction laboratory, all necessary in vitro maturation (IVM) and oocyte vitrification tools can be at hand. Furthermore, upon remission and child wish, the patient has multiple options for fertility restoration: ovarian tissue transplantation or embryo transfer after the insemination of vitrified/warmed oocytes. Hence, ovarian tissue oocyte-in vitro maturation (OTO-IVM) can be a valuable adjunct fertility preservation technique.

Selecting active matter according to motility in an acoustofluidic setup: self-propelled particles and sperm cells.

Active systems - including sperm cells, living organisms like bacteria, fish, birds, or active soft matter systems like synthetic "microswimmers" - are characterized by motility, i.e., the ability to propel using their own "engine". Motility is the key feature that distinguishes active systems from passive or externally driven systems. In a large ensemble, motility of individual species can vary in a wide range. Selecting active species according to their motility represents an exciting and challenging problem. We propose a new method for selecting active species based on their motility using an acoustofluidic setup where highly motile species escape from the acoustic trap. This is demonstrated in simulations and in experiments with self-propelled Janus particles and human sperm. The immediate application of this method is selecting highly motile sperm for medically assisted reproduction (MAR). Due to the tunable acoustic trap, the proposed method is more flexible than the existing passive microfluidic methods. The proposed selection method based on motility can also be applied to other active systems that require selecting highly motile species or removing immotile species.

Electronic witnessing in the medically assisted reproduction laboratory: insights and considerations after 10 years of use.

STUDY QUESTION: What have we learnt after 10 years of electronic witnessing? SUMMARY ANSWER: When applied correctly, an electronic witnessing system can replace manual witnessing in the medically assisted reproduction lab to prevent sample mix-up. WHAT IS KNOWN ALREADY: Electronic witnessing systems have been implemented to improve the correct identification, processing, and traceability of biological materials. When non-matching samples are simultaneously present in a single workstation, a mismatch event is generated to prevent sample mix-up. STUDY DESIGN, SIZE, DURATION: This evaluation investigates the mismatch and administrator assign rate over a 10-year period (March 2011-December 2021) with the use of an electronic witnessing system. Radiofrequency identification tags and barcodes were used for patient and sample identification. Since 2011, IVF and ICSI cycles and frozen embryo transfer cycles (FET) were included; IUIs cycles were included since 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS: The total number of tags and witnessing points were recorded. Witnessing points in a particular electronic witnessing system represent all the actions that have been performed from gamete collection through embryo production, to cryopreservation and transfer. Mismatches and administrator assigns were collected and stratified per procedure (sperm preparation, oocyte retrieval, IVF/ICSI, cleavage stage embryo or blastocyst embryo biopsy, vitrification and warming, embryo transfer, medium changeover, and IUI). Critical mismatches (such as mislabelling or non-matching samples within one work area) and critical administrator assigns (such as samples not identified by the electronic witnessing system and unconfirmed witnessing points) were selected. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 109 655 cycles were included: 53 023 IVF/ICSI, 36 347 FET, and 20 285 IUI cycles. The 724 096 used tags, led to a total of 849 650 witnessing points. The overall mismatch rate was 0.251% (2132/849 650) per witnessing point and 1.944% per cycle. In total, 144 critical mismatches occurred over the different procedures. The yearly mean critical mismatch rate was 0.017 ± 0.007% per witnessing point and 0.129 ± 0.052% per cycle. The overall administrator assign rate was 0.111% (940/849 650) per witnessing point and 0.857% per cycle, including 320 critical administrator assigns. The yearly mean critical administrator assign rate was 0.039 ± 0.010% per witnessing point and 0.301 ± 0.069% per cycle. Overall mismatch and administrator assign rates remained fairly stable during the evaluated time period. Sperm preparation and IVF/ICSI were the procedures most prone to critical mismatch and administrator assigns. LIMITATIONS, REASONS FOR CAUTION: The procedures and methods of integration of an electronic witnessing system may vary from one laboratory to another and result in differences in the potential risks related to sample identification. Individual embryos cannot (yet) be identified by such a system; this makes extra manual witnessing indispensable at certain critical steps where potential errors are not recorded. The electronic witnessing system still needs to be used in combination with manual labelling of both the bottom and lid of dishes and tubes to guarantee correct assignment in case of malfunction or incorrect use of radiofrequency identification tags. WIDER IMPLICATIONS OF THE FINDINGS: Electronic witnessing is considered to be the ultimate tool to safeguard correct identification of gametes and embryos. But this is only possible when used correctly, and proper training and attention of the staff is required. It may also induce new risks, i.e. blind witnessing of samples by the operator. STUDY FUNDING/COMPETING INTEREST(S): No funding was either sought or obtained for this study. J.S. presents webinars on RIW for CooperSurgical. The remaining authors have nothing to declare. TRIAL REGISTRATION NUMBER: N/A.

Undetectable viral RNA in follicular fluid, cumulus cells, and endometrial tissue samples in SARS-CoV-2-positive women.

OBJECTIVE: To study the presence of viral RNA in the follicular fluid, cumulus cells, and endometrial tissue samples in SARS-CoV-2-positive women undergoing assisted reproductive technology (ART). DESIGN: Prospective, single-center, observational study. SETTING: Tertiary hospital. PATIENT(S): A total of 16 patients undergoing transvaginal oocyte retrieval who had a positive SARS-CoV-2 RNA test <48 hours before the procedure. All patients underwent the retrieval between September 2020 and June 2021 and used in vitro fertilization or intracytoplasmic sperm injection. All embryos were vitrified to avoid conception during SARS-CoV-2 infection. INTERVENTION(S): Follicular fluid aspirated during oocyte retrieval, cumulus cells, and endometrial samples were analyzed for SARS-CoV-2 RNA using the RealStar SARS-CoV-2 RT-PCR-Kit1.0. MAIN OUTCOME MEASURE(S): The primary outcome parameter was the detection of viral RNA in the follicular fluid, cumulus cells, and endometrial cells. Fertilization rate, embryo developmental potential, and clinical outcome after frozen embryo transfer were secondary outcome parameters. RESULT(S): Samples from 16 patients were analyzed. Cycle threshold values of <40 were considered positive. All samples were negative for SARS-CoV-2 viral RNA. No inflammatory lesions of the endometrium were identified histologically. Fertilization rate, embryo development, and clinical outcomes after embryo transfer were reassuring. CONCLUSION(S): In women infected with SARS-CoV-2 who underwent ART, viral RNA was undetectable in the follicular fluid, cumulus cells, and endometrium. Caution is warranted in view of the small sample size, and the risk of SARS-CoV-2 affecting the embryo via ART cannot be ruled out. Adequate counseling of women and couples undergoing ART is crucial in parallel with further research on the effect of exposure of the early human embryo to SARS-CoV-2. CLINICAL TRIAL REGISTRATION NUMBER: NCT04425317.

Effect of A23187 ionophore treatment on human blastocyst development-a sibling oocyte study.

PURPOSE: To investigate whether treatment with commercially available ready-to-use A23187 ionophore (GM508-CultActive) improves embryo development outcome in patients with a history of embryo developmental problems. METHODS: This is a uni-center prospective study in which sibling oocytes of patients with embryos of poor quality on day 5 in the previous cycle were treated or not with CultActive. RESULTS: Two hundred forty-seven metaphase II (MII) oocytes from 19 cycles performed between 2016 and 2019 were included in the study. After ICSI, the sibling oocytes were assigned to the treatment group or to the control group, following an electronically generated randomization list. A number of 122 MII were treated with CultActive and 125 MII had no treatment and were assigned to the control group. No difference in fertilization rate (p = 0.255) or in the capacity of embryos to reach good quality on day 5 (p = 0.197) was observed between the two groups. The utilization rates defined as the number of embryos transferred or cryopreserved per mature oocyte (p = 0.438) or per fertilized oocytes (p = 0.299) were not significantly different between the treated group and the control group. CONCLUSION: The results of the current study do not support the use of CultActive in cases with embryo developmental problems.

Live births following fertility preservation using in-vitro maturation of ovarian tissue oocytes.

STUDY QUESTION: Can oocytes extracted from excised ovarian tissue and matured in vitro be a useful adjunct for urgent fertility preservation (FP)? SUMMARY ANSWER: Ovarian tissue oocyte in-vitro maturation (OTO-IVM) in combination with ovarian tissue cryopreservation (OTC) is a valuable adjunct technique for FP. WHAT IS KNOWN ALREADY: Despite the impressive progress in the field, options for FP for cancer patients are still limited and, depending on the technique, clinical outcome data are still scarce. STUDY DESIGN, SIZE, DURATION: This was a retrospective cohort study conducted at a university hospital-affiliated fertility clinic between January 2012 and May 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study included 77 patients who underwent unilateral oophorectomy for OTC. Cumulus-oocyte complexes (COCs) obtained during ovarian tissue processing were matured in vitro for 28-42 h. Oocytes reaching metaphase II stage were vitrified or inseminated for embryo vitrification. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, 1220 COCs were collected. The mean oocyte maturation rate was 39% ± 23% (SD). There were 64 patients who had vitrification of oocytes (6.7 ± 6.3 oocytes per patient). There were 13 patients who had ICSI of mature oocytes after IVM, with 2.0 ± 2.0 embryos vitrified per patient. Twelve patients have returned to the clinic with a desire for pregnancy. For seven of these, OTO-IVM material was thawed. Two patients had OTO-IVM oocytes warmed, with survival rates of 86% and 60%. After ICSI, six oocytes were fertilised in total, generating three good quality embryos for transfer, leading to a healthy live birth for one patient. In five patients, for whom a mean of 2.0 ± 0.8 (SD) embryos had been vitrified, seven embryos were warmed in total: one embryo did not survive the warming process; two tested genetically unsuitable for transfer; and four were transferred in separate cycles to three different patients, resulting in two healthy babies. In this small series, the live birth rate per patient after OTO-IVM, ICSI and embryo transfer was 43%. LIMITATIONS, REASONS FOR CAUTION: The retrospective study design and the limited sample size should be considered when interpreting results. WIDER IMPLICATIONS OF THE FINDINGS: The results of the study illustrate the added value of OTO-IVM in combination with OTC. We report the first live birth following the use of this appended technique combined with oocyte vitrification. STUDY FUNDING/COMPETING INTEREST(S): No external funding was used for this study. M.D.V. reports honoraria for lectures in the last 2 years from MSD and Ferring, outside the submitted work, as well as grant support from MSD. The other authors have nothing to declare. TRIAL REGISTRATION NUMBER: N/A.

Obstetric and neonatal outcome following ICSI with assisted oocyte activation by calcium ionophore treatment.

PURPOSE: Calcium ionophore treatment is being used in assisted reproductive technology (ART) for cases with previous low fertilization rate or total absence of fertilization after insemination by intracytoplasmic sperm injection or when a specific indication such as globozoospermia is present. As this technique is more invasive and differs from the physiological process of fertilization, a thorough investigation of the health of the children born following this procedure is required. We intent to report the medical outcome of all children conceived following calcium ionophore treatment in our IVF center. METHODS: One-armed descriptive study is performed to report the obstetrical and neonatal outcome of children born after using calcium ionophore treatment during the intracytoplasmic sperm injection procedure in our center. RESULTS: A number of 237 cycles were included in this study, with 74 pregnancies reported, from which 47 children (31 singletons and 16 twin children) were born. No major malformations were detected in singletons. In twins, three children were diagnosed with major malformations. Minor malformations were present in seven singletons and in one twin. CONCLUSIONS: In conclusion, our results regarding birth characteristics and congenital malformations are within the expected range but, although reassuring, should be interpreted with caution due to the small number of children included.

In vitro maturation (IVM) of oocytes recovered from ovariectomy specimens in the laboratory: a promising "ex vivo" method of oocyte cryopreservation resulting in the first report of an ongoing pregnancy in Europe.

PURPOSE: We present our center's experience with 34 consecutive cases who underwent in vitro maturation (IVM) of oocytes obtained from ovariectomy specimens and compare our data with updated literature data. METHODS: Feasibility and efficiency of oocyte collection during ovarian tissue processing was assessed by the recovery rate, maturation rate, and embryological development after IVM. RESULTS: On average, 14 immature oocytes were retrieved per patient during ovarian tissue processing in 33/34 patients. The overall maturation rate after IVM was 36%. The maturation rate correlated with the age of the patient and the duration of IVM. Predominately, oocyte vitrification was performed. Eight couples preferred embryo cryopreservation. Here, a 65% fertilization rate was obtained and at least one good-quality day 3 embryo was cryopreserved in 7/8 couples. The retrieval of oocytes ex vivo resulted in mature oocytes or embryos available for vitrification in 79% of patients. One patient with ovarian insufficiency following therapeutic embolization of the left uterine and the right ovarian artery because of an arteriovenous malformation had an embryo transfer of one good-quality warmed embryo generated after IVM ex vivo, which resulted in an ongoing clinical pregnancy. CONCLUSIONS: IVM of oocytes obtained ex vivo during the processing of ovarian cortex prior to cryopreservation is a procedure with emerging promise for patients at risk for fertility loss, as illustrated by the reported pregnancy. However, more data are needed in order to estimate the overall success rate and safety of this novel approach.

Establishment of hESC lines from the inner cell mass of blastocyst-stage embryos and single blastomeres of 4-cell stage embryos.

More than 600 human embryonic stem cell (hESC) lines have been reported today at the human European Embryonic Stem Cell Registry ( http://www.hescreg.eu/ ). Despite these high numbers, there are currently no general protocols for derivation, culture, and characterization of hESC. Moreover, data on the culture of the embryo used for the derivation (medium, day of ICM isolation) are usually not available but can have an impact on the derivation rate. We present here the protocols for derivation, culture and characterization as we applied them for the 22 hESC lines (named VUB-hESC) in our laboratory.

HLA-G expression in human embryonic stem cells and preimplantation embryos.

Human leukocyte Ag-G, a tolerogenic molecule that acts on cells of both innate and adaptive immunity, plays an important role in tumor progression, transplantation, placentation, as well as the protection of the allogeneic fetus from the maternal immune system. We investigated HLA-G mRNA and protein expression in human embryonic stem cells (hESC) derived from the inner cell mass (ICM) of blastocysts. hESC self-renew indefinitely in culture while maintaining pluripotency, providing an unlimited source of cells for therapy. HLA-G mRNA was present in early and late passage hESC, as assessed by real time RT-PCR. Protein expression was demonstrated by flow cytometry, immunocytochemistry, and ELISA on an hESC extract. Binding of HLA-G with its ILT2 receptor demonstrated the functional active status. To verify this finding in a physiologically relevant setting, HLA-G protein expression was investigated during preimplantation development. We demonstrated HLA-G protein expression in oocytes, cleavage stage embryos, and blastocysts, where we find it in trophectoderms but also in ICM cells. During blastocyst development, a downregulation of HLA-G in the ICM cells was present. This data might be important for cell therapy and transplantation because undifferentiated hESC can contaminate the transplant of differentiated stem cells and develop into malignant cancer cells.

Derivation, culture, and characterization of VUB hESC lines.

In this report, we present the derivation and characterization of 15 hESC lines established at the Vrije Universiteit Brussel, Belgium in collaboration with the Universitair Ziekenhuis Brussel, Belgium, using surplus in vitro fertilization embryos and embryos carrying monogenic disorders donated for research. Four lines were derived from blastocyst-stage embryos presumed to be genetically normal, and 11 hESC lines were obtained from embryos shown to carry genetic mutations by preimplantation genetic diagnosis. All the lines express markers of pluripotency as determined by immunocytochemistry and RT-PCR, and formed teratomas when injected into SCID mice. All VUB hESC lines, except for VUB17, are reported in the European hESC registry and are available upon request after signing a Material Transfer Agreement from the VUB (contact person: Prof. Dr. Karen Sermon; Karen.Sermon@uzbrussel.be).

Noggin, retinoids, and fibroblast growth factor regulate hepatic or pancreatic fate of human embryonic stem cells.

BACKGROUND & AIMS: New sources of beta cells are needed to develop cell therapies for patients with diabetes. An in vitro, sequential method has been developed to derive pancreatic progenitors, but this technique has not been used for other cell lines. We investigated whether definitive endoderm derived from human embryonic stem (hES) cells might be used to create beta cells. METHODS: Five hES cell lines were induced to form pancreatic progenitors and analyzed for pancreas markers. Cells were incubated with a bone morphogenetic protein (BMP) antagonist, retinoids, a Hedgehog antagonist, or fibroblast growth factor (FGF) and phenotypes were analyzed. RESULTS: Four hES cell lines sequentially generated definitive endoderm, primitive gut, and posterior foregut equivalents, as described previously. However, functional hepatocytes, rather than pancreas progenitors, developed. Consistent with liver development, FGF and BMP signaling pathways were involved in this process; their inhibition disrupted hepatocyte differentiation. During early stages of development, exposure of cells to noggin and retinoid acid, followed by FGF10, generated pancreatic cells (PDX1+; 50%-80%) that coexpressed FOXA2, HNF6, and SOX9. CONCLUSIONS: These findings demonstrate the combined functions of endogenous BMP and supplemented FGF in inducing differentiation of hepatocytes from hES cells and the ability to shift developmental pathways from hepatic to pancreatic cell differentiation. Although additional signals appear to be required for full specification of PDX1(+) early pancreatic progenitors (via PTF1a and NKX6.1 coexpression), these findings indicate the signaling pathways required for differentiation of bipotential progenitors.

Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos.

BACKGROUND: Recently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres. METHODS: Four 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs). RESULTS: Ten out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT-PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype. CONCLUSION: We report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent.

Recurrent chromosomal abnormalities in human embryonic stem cells.

Cultured human embryonic stem (hES) cells have a known predisposition to aneuploidy of chromosomes 12, 17 and X. We studied 17 hES cell lines by array-based comparative genomic hybridization (aCGH) and found that the cells accumulate other recurrent chromosomal abnormalities, including amplification at 20q11.21 and a derivative chromosome 18. These genomic changes have a variable impact at the transcriptional level.

Efficient differentiation of human embryonic stem cells into a homogeneous population of osteoprogenitor-like cells.

The use of human embryonic stem cells (hESC) in both research and therapeutic applications requires relatively large homogeneous populations of differentiated cells. The differentiation of three hESC lines into highly homogeneous populations of osteoprogenitor-like (hESC-OPL) cells is reported here. These cells could be expanded in a defined culture system for more than 18 passages, and showed a fibroblast-like morphology and a normal stable karyotype. The cells were strongly positive for the same antigenic markers as mesenchymal stem cells but negative for markers of haematopoetic stem cells. The hESC-OPL cells were able to differentiate into the osteogenic, but not into the chondrogenic or adipogenic, lineage and were positive for markers of early stages of osteogenic differentiation. When cultured in the presence of osteogenic supplements, the cells indicated the capacity to achieve, under inductive conditions, a mature osteoblast phenotype. The differentiation protocol is based on a monolayer approach, and does not require any exogenous factors other than fetal calf serum, or coculture systems of animal or human origin. This method is likely to be amenable to large-scale production of homogeneous osteoprogenitor-like cells and thus overcomes one of the major problems of differentiation of hESC, with important relevance for further cell therapy studies.

Selection of reference genes in mouse embryos and in differentiating human and mouse ES cells.

Embryonic Stem (ES) cells have the potential to form every cell of the body and thus are of great promise for tissue transplantation. One of the rising techniques that allows studying the differentiation state of ES cells is quantitative RT-PCR (qRT-PCR). When relative quantification by qRT-PCR is applied, accurate normalization is necessary, since differentiated embryonic stem cells and developing embryos contain heterogeneous cell populations. Corrections for variations in the qRT-PCR reaction are needed to allow comparisons between different samples. We applied the normalization tools geNorm and Normfinder to ten reference genes identifying the most stable ones for relative quantification of gene expression during differentiation of human ES cells, as well as in differentiated mouse ES cells and in the developing mouse embryo. For relative quantification by qRT-PCR in these systems, we advise to use normalization factors based on multiple stable reference genes. However, when the use of several reference genes would be unpractical, a single reference gene in each experimental setup could be sufficient. When looking for single stable reference genes, beta-actin works best in both mouse embryo and ES cell experiments and glyceraldehyde-3-phosphate-dehydrogenase can be applied in both mouse and human ES cell experiments.