Importance of real-time measurement of sperm head morphology in intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is an important technique in male infertility treatment. Currently, sperm selection for ICSI in human assisted reproductive technology (ART) is subjective, based on a visual assessment by the operator. Therefore, it is desirable to develop methods that can objectively provide an accurate assessment of the shape and size of sperm heads that use low-magnification microscopy available in most standard fertility clinics. Recent studies have shown a correlation between sperm head size and shape and chromosomal abnormalities, and fertilization rate, and various attempts have been made to establish automated computer-based measurement of the sperm head itself. For example, a dictionary-learning technique and a deep-learning-based method have both been developed. Recently, an automatic algorithm was reported that detects sperm head malformations in real time for selection of the best sperm for ICSI. These data suggest that a real-time sperm selection system for use in ICSI is necessary. Moreover, these systems should incorporate inverted microscopes (×400-600 magnification) but not the fluorescence microscopy techniques often used for a dictionary-learning technique and a deep-learning-based method. These advances are expected to improve future success rates of ARTs. In this review, we summarize recent reports on the assessment of sperm head shape, size, and acrosome status in relation to fertility, and propose further improvements that can be made to the ARTs used in infertility treatments.

Correlation between the pronucleus size and the potential for human single pronucleus zygotes to develop into blastocysts : 1PN zygotes with large pronuclei can expect an embryo development to the blastocyst stage that is similar to the development of 2PN zygotes.

PURPOSE: In this study, we examined the correlation between pronucleus size and the potential for human single pronucleus (1PN) zygotes to develop into blastocysts after IVF and ICSI. METHODS: This study included 112 patients who underwent a total of 112 cycles of IVF/ICSI. To evaluate embryo development, 1PN zygotes were compared with 2PN zygotes in the same IVF/ICSI cycle (control cycles) using time-lapse live embryo imaging. To assess the potential for blastocyst formation, cutoff values for pronuclear area and diameter were established through receiver operating characteristic curve analysis, after which 1PN zygotes were classified based on those cutoff values. RESULTS: Among 1PN zygotes cultured to day 5/6, the rate of embryo development was significantly lower than from 2PN zygotes. However, the rates of blastocyst formation and good quality blastocysts from 1PN zygotes with large pronuclear areas (≥ 710 µm2) or diameters (≥ 31 µm) were significantly higher than from 1PN zygotes with smaller pronuclear areas (≤ 509, 510-609, and 610-709 µm2) or diameters (≤ 24, 25-27,and 28-30 µm) (P < 0.01). Moreover, the results for 1PN zygotes with large pronuclei were similar to those for 2PN zygotes. CONCLUSIONS: The developmental potential of 1PN zygotes with large pronuclear areas (≥ 710 µm2) or diameters (31 µm) appears to be similar to that of 2PN zygotes, and measurement of pronuclear area or diameter in 1PN zygotes is a simple, potentially useful, clinical method.

The normality of sperm in an infertile man with ring chromosome 15: a case report.

PURPOSE: The purpose of this report is to analyze the chromosome status and fertilization capability of sperm obtained from an infertile male patient with ring chromosome 15. METHODS: This was a case report at a private in vitro fertilization clinic. A man diagnosed with severe oligozoospermia carrying ring chromosome 15. To evaluate the chromosome status and fertilization capability, sperm from a patient carrying ring chromosome 15 were injected into enucleated mouse oocytes. RESULTS: The karyotypes of motile sperm from a patient carrying ring chromosome 15 were normal, and ring chromosome 15 was not observed in the chromosome spread samples of 1PN. In addition, these motile sperm retained the fertilization capability. However, the fertilization rates decreased (85.2, 76.2, and 64.3%, respectively) along with the decline of the aspect ratio of the sperm head (≥ 1.50, 1.30-1.49, and < 1.30, respectively). CONCLUSIONS: The karyotypes were normal without ring chromosome 15, and motile sperm with a high aspect ratio showed adequate potential for fertilization.

Clinical outcomes after IVF or ICSI using human blastocysts derived from oocytes containing aggregates of smooth endoplasmic reticulum.

In this study the clinical and neo-natal outcomes after transfer of blastocysts derived from oocytes containing aggregates of smooth endoplasmic reticulum (SER) were compared between IVF and intracytoplasmic sperm injection (ICSI) cycles. Clinical and neo-natal outcomes of blastocysts in cycles with at least one SER metaphase II oocyte (SER + MII; SER + cycles) did not significantly differ between the two insemination methods. When SER + MII were cultured to day 5/6, fertilization, embryo cleavage and blastocyst rates were not significantly different between IVF and ICSI cycles. In vitrified-warmed blastocyst transfer cycles, the clinical pregnancy rates from SER + MII in IVF and ICSI did not significantly differ. In this study, 52 blastocysts (27 IVF and 25 ICSI) derived from SER + MII were transferred, yielding 15 newborns (5 IVF and 10 ICSI) and no malformations. Moreover, 300 blastocysts (175 IVF and 125 ICSI) derived from SER-MII were transferred, yielding 55 newborns (24 IVF and 31 ICSI cycles). Thus, blastocysts derived from SER + cycles exhibited an acceptable ongoing pregnancy rate after IVF (n = 125) or ICSI (n = 117) cycles. In conclusion, blastocysts from SER + MII in both IVF and ICSI cycles yield adequate ongoing pregnancy rates with neo-natal outcomes that do not differ from SER-MII.

Embryological outcomes in cycles with human oocytes containing large tubular smooth endoplasmic reticulum clusters after conventional in vitro fertilization.

There have been no studies analyzing the effect of large aggregates of tubular smooth endoplasmic reticulum (aSERT) after conventional in vitro fertilization (cIVF). The aim of this study was to investigate whether aSERT can be identified after cIVF and the association between the embryological outcomes of oocytes in cycles with aSERT. This is a retrospective study examining embryological data from cIVF cycles showing the presence of aSERT in oocytes 5-6 h after cIVF. To evaluate embryo quality, cIVF cycles with at least one aSERT-metaphase II (MII) oocyte observed (cycles with aSERT) were compared to cycles with normal-MII oocytes (control cycles). Among the 4098 MII oocytes observed in 579 cycles, aSERT was detected in 100 MII oocytes in 51 cycles (8.8%). The fertilization rate, the rate of embryo development on day 3 and day 5-6 did not significantly differ between cycles with aSERT and control group. However, aSERT-MII oocytes had lower rates for both blastocysts and good quality blastocysts (p < 0.05). aSERT can be detected in the cytoplasm by removing the cumulus cell 5 h after cIVF. However, aSERT-MII oocytes do not affect other normal-MII oocytes in cycles with aSERT.

A Simple Method for Transportation of Mouse Embryos Using Microtubes and a Warm Box.

Generally, transportation of preimplantation embryos without freezing requires incubators that can maintain an optimal culture environment with a suitable gas phase, temperature, and humidity. Such incubators are expensive to transport. We reported previously that normal offspring were obtained when the gas phase and temperature could be maintained during transportation. However, that system used plastic dishes for embryo culture and is unsuitable for long-distance transport of live embryos. Here, we developed a simple low-cost embryo transportation system. Instead of plastic dishes, several types of microtubes-usually used for molecular analysis-were tested for embryo culture. When they were washed and attached to a gas-permeable film, the rate of embryo development from the 1-cell to blastocyst stage was more than 90%. The quality of these blastocysts and the rate of full-term development after embryo transfer to recipient female mice were similar to those of a dish-cultured control group. Next, we developed a small warm box powered by a battery instead of mains power, which could maintain an optimal temperature for embryo development during transport. When 1-cell embryos derived from BDF1, C57BL/6, C3H/He and ICR mouse strains were transported by a parcel-delivery service over 3 days using microtubes and the box, they developed to blastocysts with rates similar to controls. After the embryos had been transferred into recipient female mice, healthy offspring were obtained without any losses except for the C3H/He strain. Thus, transport of mouse embryos is possible using this very simple method, which might prove useful in the field of reproductive medicine.

Birth of nine normal healthy babies following transfer of blastocysts derived from human single-pronucleate zygotes.

PURPOSE: The purpose of this study is to examine the clinical outcomes of blastocysts derived from human single-pronucleate (1PN) embryos after conventional in vitro fertilization (cIVF) and intracytoplasmic sperm injection (ICSI) cycles. METHODS: This was a retrospective study at a reproductive center of a hospital. To evaluate embryo quality and clinical outcomes, cIVF or ICSI cycles with one or more 1PN embryos were compared with same cycles with 2PN embryos (control cycles). RESULTS: A total of 623 cycles (426 cIVF cycles and 197 ICSI cycles) were treated with cIVF or ICSI. The single pronuclear status rate was similar between cIVF (22.1 %) and ICSI (25.1 %) cycles. Although the development rates of 1PN embryos on day 3 and day 5/6 in cIVF were significantly higher than those in ICSI, those of 1PN embryos in cIVF were significantly lower compared to 2PN embryos (p < 0.01). Nonetheless, the ongoing pregnancy rates achieved with 1PN blastocysts in 1PN embryos did not significantly differ from the control group. Thirty-three transfer cycles with 33 blastocysts derived from 1PN embryos in cIVF resulted in nine deliveries with no newborn malformations; however, no implantation was observed in three ICSI cycles. CONCLUSION: Although the blastocyst formation rate of 1PN embryos was significantly lower than 2PN embryos in cIVF and ICSI cycles, 1PN blastocysts in cIVF, and not from ICSI, demonstrated an adequate ongoing pregnancy rate. These results suggested that 1PN blastocysts in cIVF are available for clinical use and may lead to an increase in the chance of pregnancy in patients receiving assisted reproductive technology with 1PN embryos.

Latrunculin A treatment prevents abnormal chromosome segregation for successful development of cloned embryos.

Somatic cell nuclear transfer to an enucleated oocyte is used for reprogramming somatic cells with the aim of achieving totipotency, but most cloned embryos die in the uterus after transfer. While modifying epigenetic states of cloned embryos can improve their development, the production rate of cloned embryos can also be enhanced by changing other factors. It has already been shown that abnormal chromosome segregation (ACS) is a major cause of the developmental failure of cloned embryos and that Latrunculin A (LatA), an actin polymerization inhibitor, improves F-actin formation and birth rate of cloned embryos. Since F-actin is important for chromosome congression in embryos, here we examined the relation between ACS and F-actin in cloned embryos. Using LatA treatment, the occurrence of ACS decreased significantly whereas cloned embryo-specific epigenetic abnormalities such as dimethylation of histone H3 at lysine 9 (H3K9me2) could not be corrected. In contrast, when H3K9me2 was normalized using the G9a histone methyltransferase inhibitor BIX-01294, the Magea2 gene-essential for normal development but never before expressed in cloned embryos-was expressed. However, this did not increase the cloning success rate. Thus, non-epigenetic factors also play an important role in determining the efficiency of mouse cloning.

Failure of GnRH agonist-triggered oocyte maturation: its cause and management.

OBJECTIVE: In the present study we investigated the cause of GnRHa's failure to trigger oocyte maturation and present an effective rescue protocol for use when failure occurs. DESIGN: Case reports. SETTING: One in vitro fertilization (IVF) center. PATIENT(S): Eight cases of failure of GnRH agonist (GnRHa)-triggered oocyte maturation and one case of GnRHa successfully triggered oocyte maturation, despite a weak response in the LH-RH test. MAIN OUTCOME MEASURE(S): All cases were successfully rescued by re-triggering maturation using HCG, with seven of the eight patients achieving ongoing pregnancy and successful delivery. RESULT(S): In three patients the cause of the oocyte maturation failure was likely temporal or continuous severe down-regulation of the hypothalamus-pituitary-axis, the cause was unknown in the other five patients. CONCLUSION(S): In cases where GnRHa fails to trigger oocyte maturation, a rescue protocol entailing re-triggering with HCG can produce successful outcomes.

Offspring from mouse embryos developed using a simple incubator-free culture system with a deoxidizing agent.

To culture preimplantation embryos in vitro, water-jacketed CO(2) incubators are used widely for maintaining an optimal culture environment in terms of gas phase, temperature and humidity. We investigated the possibility of mouse embryo culture in a plastic bag kept at 37°C. Zygotes derived from in vitro fertilization or collected from naturally mated B6D2F1 female mice were put in a drop of medium on a plastic culture dish and then placed in a commercially available plastic bag. When these were placed in an oven under air at 37°C for 96 h, the rate of blastocyst development and the cell numbers of embryos decreased. However, when the concentration of O(2) was reduced to 5% using a deoxidizing agent and a small oxygen meter, most zygotes developed into blastocysts. These blastocysts were judged normal according to their cell number, Oct3/4 and Cdx2 gene expression levels, the apoptosis rate and the potential for full-term development after embryo transfer to pseudopregnant recipients. Furthermore, using this system, normal offspring were obtained simply by keeping the bag on a warming plate. This culture method was applied successfully to both hybrid and inbred strains. In addition, because the developing embryos could be observed through the transparent wall of the bag, it was possible to capture time-lapse images of live embryos until the blastocyst stage without needing an expensive microscope-based incubation chamber. These results suggest that mouse zygotes are more resilient to their environment than generally believed. This method might prove useful in economical culture systems or for the international shipment of embryos.