A novel solution for freezing small numbers of spermatozoa using a sperm vitrification device.

STUDY QUESTION: Does a novel sperm vitrification device (SpermVD) provide an efficient method for freezing a small number of human spermatozoa from men suffering from non-obstructive azoospermia? SUMMARY ANSWER: The novel SpermVD is an efficient and simple carrier method for freezing a small number of spermatozoa in low-volume droplets, reducing post-thaw search time from hours to minutes, allowing a 96% recovery rate and leading to successful use of sperm for fertilization. WHAT IS KNOWN ALREADY: Previous methods for cryopreservation of small numbers of human spermatozoa (e.g. mini-straws, ICSI pipette, alginate beads, cryoloop) have been proposed as a solution for cases of severe male infertility. Many drawbacks have prevented their widespread use, including cumbersome preparation and sperm retrieval procedures, and the fact that the thawed spermatozoa are not immediately available for micromanipulation and required additional treatment which posed excess risk of harm. STUDY DESIGN, SIZE, DURATION: We conducted a feasibility experiment of the novel SpermVD and a prospective cohort study of ICSI cycles in men suffering from non-obstructive azoospermia in two outpatient fertility IVF clinics, from 2015 through 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients underwent extended ejaculate search prior to the day of oocyte retrieval, and any single motile spermatozoa found was transferred to 0.8 µl droplets of 1:1 washing medium/cryoprotectant on the SpermVD, then plunged into liquid nitrogen for cryopreservation. In patients with non-obstructive azoospermia who underwent surgical TESE, both the motile and immotile spermatozoa found underwent cryopreservation using the SpermVD. On the day of oocyte retrieval, the SpermVD was thawed, directly transfered to the ICSI plate and retrieved spermatozoa were used for the ICSI procedure. MAIN RESULTS AND THE ROLE OF CHANCE: The prospective cohort included 44 cases. We used the SpermVD to vitrify 631 spermatozoa, of which 540 (86%) were motile. The average number of frozen spermatozoa per patient was 14.3 ± 9.3. After thawing, we retrieved 607 spermatozoa, producing a recovery rate of 96%. The average number of thawed spermatozoa was 13.8 ± 9.2. The recovery of 180 thawed motile sperm accounted for 33% of all frozen motile spermatozoa. The fertilization rate was 59%. Of 44 oocyte retrieval procedures, 24 (55%) clinical pregnancies were achieved. The delivery rate (not including three ongoing pregnancies) was 32% and the miscarriage rate was 29%. LIMITATIONS, REASONS FOR CAUTION: Although we presented the SpermVD on 44 cases, a larger cohort would provide more information. Moreover, we cryopreserved only motile sperm from the ejaculates and not immotile sperm, thus limiting the knowledge regarding the efficacy of the VD for immotile sperm from this source. WIDER IMPLICATIONS OF THE FINDINGS: The novel SpermVD is a simple efficient carrier, optimizing the protocol for freezing a small number of spermatozoa. It may allow for the routine use of frozen spermatozoa after TESE for men suffering from non-obstructive azoospermia and thus avoid repeated TESE surgeries. Furthermore, in cases of non-obstructive azoospermia, routine cryopreservation of the retrieved spermatozoa prior to the IVF cycle may avoid the risk of cycle cancelation and thus decrease the number of unnecessary oocyte retrieval procedures. STUDY FUNDING/COMPETING INTEREST(S): There was no external funding. There are no competing interests. TRIAL REGISTRATION NUMBER: IRB no 00119-16-ASMC.

Fertility outcomes after extended searches for ejaculated spermatozoa in men with virtual azoospermia.

OBJECTIVE: To assess the fertility outcomes of extended searches for ejaculated spermatozoa in men with virtual azoospermia. DESIGN: A retrospective cohort of 242 couples whose male partner suffered from nonobstructive azoospermia and who were treated with the use of intracytoplasmic sperm injection (ICSI). SETTING: Not applicable. PATIENT(S): One hundred forty patients were referred to an extended search in the ejaculate and 102 patients were referred to microsurgical testicular sperm extraction (microTESE). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Rates of sperm retrieval, fertilization, and pregnancy, take-home baby rate, and missed abortion rate were analyzed and compared. RESULT(S): In the ejaculated spermatozoa group, motile spermatozoa were retrieved in 91 cases (65%) and on oocyte pick-up day in 71 cases (78%), compared with 70 cases (68%) in the microTESE group, with a similar incidence of sperm retrieval between groups. No significant difference was found between groups regarding mean number of embryo transfer and fertilization and pregnancy rates. There was no significant difference between groups regarding take-home baby rate. A significantly higher first-trimester missed abortion rate was found in the ejaculated sperm group (n = 14; 52%) compared with the microTESE group (n = 3; 8.6%). CONCLUSION(S): Conducting an extended spermatozoa search in the ejaculate of men with virtual azoospermia can provide pregnancy rates similar to those obtained with the use of microTESE, with a higher rate of spontaneous abortions in the ejaculate group.

Intracytoplasmic morphologically selected sperm injection versus intracytoplasmic sperm injection: a step toward a clinical algorithm.

OBJECTIVE: To study the advantage of intracytoplasmic morphologically selected sperm injection (IMSI) versus intracytoplasmic sperm injection (ICSI) in the first artificial reproductive technology (ART) cycle and in consecutive cycles. DESIGN: A cohort study. SETTING: Single outpatient fertility center. PATIENT(S): Couples presenting with male factor infertility, requiring ovum micromanipulation. INTERVENTION(S): The ICSI or IMSI was performed according to the couple's choice. MAIN OUTCOME MEASURE(S): Clinical intrauterine pregnancies and deliveries. RESULT(S): A total of 1,891 IVF-ICSI cycles and 577 IVF-IMSI cycles were included. In the first IVF treatment, pregnancy rates (PRs) were 46% and 47%, respectively, and delivery rates were 23% versus 30%, respectively. In the second cycle to follow a failed ICSI, PRs and delivery rates were significantly higher for patients who chose to shift to the IMSI technique compared with patients who chose to go through a second IVF-ICSI cycle (56% vs. 38% PRs and 28% vs. 18% delivery rates, respectively). In the following cycles a significant difference was demonstrated in both PR and delivery rates in favor of patients shifting between treatments. In a multivariate analysis an approximate threefold increased chance existed for both pregnancy and delivery only in the case of couples failing an ICSI attempt who shifted to IMSI. CONCLUSION(S): Our present experience supports refraining from repeated IMSI cycles. In light of improved PRs and delivery rates, we recommend promoting the IMSI method for couples who failed ICSI cycle, once or more.

Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension.

Undifferentiated human embryonic stem cells (hESCs) are currently propagated on a relatively small scale as monolayer colonies. Culture of hESCs as floating aggregates is widely used for induction of differentiation into embryoid bodies. Here we show that hESC lines can be derived from floating inner cell masses in suspension culture conditions that do not involve feeder cells or microcarriers. This culture system supports prolonged propagation of the pluripotent stem cells as floating clusters without their differentiation into embryoid bodies. HESCs cultivated as aggregates in suspension maintain the expression of pluripotency markers and can differentiate into progeny of the three germ layers both in vitro and in vivo. We further show the controlled differentiation of hESC clusters in suspension into neural spheres. These results pave the way for large-scale expansion and controlled differentiation of hESCs in suspension, which would be valuable in basic and applied research.

The role of FGF-signaling in early neural specification of human embryonic stem cells.

The mechanisms that govern human neural specification are not completely characterized. Here we used human embryonic stem cells (hESCs) to study the role of fibroblast growth factor (FGF)-signaling in early human neural specification. Differentiation was obtained by culturing clusters of hESCs in chemically-defined medium. We show that FGF-signaling, which is endogenously active during early differentiation of hESCs, induces early neural specification, while its blockage inhibits neuralization. The early neuralization effect of FGF-signaling is not mediated by promoting the proliferation of existing neural precursors (NPs) or prevention of their apoptosis. The neural instructive effect of FGF-signaling occurs after an initial FGF-independent differentiation into primitive ectoderm-like fate. We further show that FGF-signaling can induce neuralization by a mechanism which is independent of modulating bone morphogenic protein (BMP)-signaling. Still, FGF-signaling is not essential for hESC neuralization which can occur in the absence of FGF and BMP-signaling. Collectively, our data suggest that human neural induction is instructed by FGF-signaling, though neuralization of hESCs can occur in its absence.

Is fine morphology of the human sperm nuclei affected by in vitro incubation at 37 degrees C?

OBJECTIVE: Incubation of ejaculated spermatozoa at 37 degrees C is recommended for IVF-intracytoplasmic sperm injection. Preselection of sperm cells with morphologically normal nuclei before microinjection, adapted in our laboratory, is usually a time-consuming procedure. Therefore, we aimed to verify whether incubation at 37 degrees C could affect the morphologic integrity of sperm nuclei. DESIGN: Time-kinetics studies testing fine morphology of the sperm nuclei upon prolonged in vitro incubation. SETTING: Male Fertility Laboratory at Bar-Ilan University, Ramat Gan, Israel. PATIENT(S): Forty-two males selected at random, who were referred for sperm preselection before ICSI. MAIN OUTCOME MEASURE(S): Morphologic integrity of the sperm nuclei, obtained by motile sperm organelle morphology examination. RESULT(S): After 2 hours of incubation at 37 degrees C a significant decrease occurred in the morphologic integrity of the sperm nuclei, compared with the initial state (4.7% +/- 2.8% vs. 6.8% +/- 3.5%, t = 3.2, Por=2 hours) sperm manipulations for assisted reproduction therapy should be performed at 21 degrees C rather than 37 degrees C.

ATM-mediated response to DNA double strand breaks in human neurons derived from stem cells.

Ataxia-telangiectasia (A-T) is a multi-system genomic instability syndrome that is caused by loss or inactivation of the ATM protein kinase. ATM is largely nuclear in proliferating cells, and activates an extensive network of pathways in response to double strand breaks (DSBs) in the DNA by phosphorylating key proteins in these pathways. The prominent symptom of A-T is neuronal degeneration, making the elucidation of ATM's functions in neurons essential to understanding the disease. It has been suggested that ATM is cytoplasmic in neurons and functions in processes that are not associated with the DNA damage response. Recently we showed that in human neuron-like cells obtained by in vitro differentiation of neuroblastomas, ATM was largely nuclear and mediated the DSB response as in proliferating cells. We have now extended these studies to two additional model systems: neurons derived from human embryonic stem cells, and cortical neurons derived from neural stem cells. The results substantiate the notion that ATM is nuclear in human neurons and mediates the DSB response, the same as it does in proliferating cells. We present here unique and powerful model systems to further study the ATM-mediated network in neurons.

Lentiviral vectors harboring a dual-gene system allow high and homogeneous transgene expression in selected polyclonal human embryonic stem cells.

Genetic modification of human embryonic stem cells (hESCs) is highly valuable for their exploitation in basic science and therapeutic applications. Here we developed lentiviral vectors (LVs) constitutively expressing a reporter and a selectable marker to enable high and homogeneous transgene expression within polyclonal hESCs. LVs carrying GFP and a downstream puromycin resistance gene, linked by the encephalomyocarditis virus (EMCV) or poliovirus internal ribosome entry sites (IRES), allowed homogeneous GFP expression after antibiotic selection. The GFP-expression levels were higher with the EMCV IRES. We also developed dual-promoter vectors harboring a reporter and an antibiotic resistance gene under the regulation of human EF1alpha and PGK1 promoters, respectively. Optimal efficiency was obtained when: (1) the reporter cassette was upstream rather than downstream of the selectable marker cassette, (2) the puromycin rather than the neomycin resistance gene was used, (3) a 5' deletion (314 bp) was created in the PGK promoter, and (4) two copies of a 120-bp element derived from the hamster Aprt CpG island were introduced upstream of the EF1alpha promoter. In summary, we developed bicistronic and novel dual-promoter LVs that enable high and homogeneous expression of transgenes by polyclonal hESCs after antibiotic selection. These vectors may provide important tools for basic and applied research on hESCs.

Derivation of neural precursors from human embryonic stem cells in the presence of noggin.

The utilization of human embryonic stem cells (hESC) for basic and applied research is hampered by limitations in directing their differentiation. Empirical poorly defined methods are currently used to develop cultures enriched for distinct cell types. Here, we report the derivation of neural precursors (NPs) from hESC in a defined culture system that includes the bone morphogenetic protein antagonist noggin. When hESC are cultured as floating aggregates in defined medium and BMP signaling is repressed by noggin, non-neural differentiation is suppressed, and the cell aggregates develop into spheres highly enriched for proliferating NPs. The NPs can differentiate into astrocytes, oligodendrocytes, and mature electrophysiologically functional neurons. During prolonged propagation, the differentiation potential of the NPs shifts from neuronal to glial fate. The presented noggin-dependent controlled conversion of hESC into NPs is valuable for the study of human neurogenesis, the development of new drugs, and is an important step towards the potential utilization of hESC in neural transplantation therapy.

Stable genetic modification of human embryonic stem cells by lentiviral vectors.

Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass of the early preimplantation embryo. An efficient strategy for stable genetic modification of hES cells may be highly valuable for manipulating the cells in vitro and may promote the study of hES cell biology, human embryogenesis, and the development of cell-based therapies. Here, we demonstrate that vectors derived from self-inactivating (SIN) human immunodeficiency virus type 1 (HIV-1) are efficient tools for stable genetic modification of hES cells. Transduction of hES cells by a modified vector derived from SIN HIV-1 and containing the woodchuck hepatitis regulatory element (WPRE) and the central polypurine tract (cPPT) sequence facilitated stable transgene expression during prolonged (38 weeks) undifferentiated proliferation in vitro. Southern blot analysis revealed that the viral vector had integrated into the host cells' DNA. Transgene expression was maintained throughout differentiation into progeny of all three germ layers both in vitro and in vivo in teratomas. Thus, the transduced hES cells retained the capability for self-renewal and their pluripotent potential. Genetic modification of hES cells by lentiviral vectors provides a powerful tool for basic and applied research in the area of human ES cells.