The motility of frozen-thawed sperm is very different among not only species but also among individuals within the same species. Since the optimal protocol needs to be adjusted for each species produce good quality frozen sperm, we investigated the pretreatment, cooling, freezing and thawing methods of sperm cryopreservation. In the first part of sperm cryopreservation, we carried out either the isolation of sperm from human semen or the addition of a neutralizer to boar semen to protect the sperm from bacteria-released endotoxins. During the cooling and freezing processes, we determined the optimal combination of hyper-osmolality solution and low concentration of glycerol. Although the motility of the sperm immediately after thawing was very high after this novel freezing technique, the motility decreased in a time dependent manner due to the increase of intracellular Ca²⁺ in sperm. To suppress Ca²⁺ uptake just after thawing, we added the Ca²⁺ chelator, EGTA to the sperm thawing media, which improved the post-thawed sperm motility and sperm membrane integrity, and decreased the rate of sperm DNA fragmentation. Using the frozen-thawed boar sperm, the conception rate and the number of pups per delivery were significantly greater than those of the conventional method. This method should contribute to human infertility care.

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Ovarian primordial follicles do not regenerate and proliferate in vivo, and their number decreases with aging as well as with certain pathogeneses. When the number of residual follicles decreases to a threshold level, the initial step of follicle development, namely, the activation of dormant follicles is disturbed and subsequent follicle growth is suppressed resulting in anovulation and amenorrhea. Because the exact mechanisms of the activation of dormant primordial follicles still remain to be determined, we focused on intracellular signaling in the activation process and succeeded in activating those follicles through the activation of PI3K-Akt-Foxo3 pathway (IVA; in vitro activation). Based on the success of IVA, we performed clinical studies to generate mature oocytes from patients with primary ovarian insufficiency, who had few residual follicles in their ovaries, and we have reported successful pregnancies and a birth following IVA. In this review, I show our IVA approach and discuss future possibilities for the infertility treatment in patients with diminished ovarian reserve.

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The Time-Lapse Imaging-Incubator System, which is attracting attention in the field of reproductive medicine, is an imaging system that can perform imaging and culture at the same time and has two capabilities. The first capability is that it can be used to observe embryo development. Furthermore, more information can be obtained by analyzing embryo growth at different time points than by morphological observation at a single point in time. The second capability is that it can be used to perform embryonic observation with images direct observation with a microscope is not necessary. This system enables continuous observation and continuous culture that maintains the hypoxic environment without the need to expose embryos entirely to air in a series of culture periods during embryo growth.

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Currently, in Japan, the most commonly used embryo vitrification method is “ultra-rapid vitrification” which is designed to achieve the maximum cooling rate by exposing embryos directly to LN2 in a tiny droplet of cryoprotectant. Though this method has yielded acceptable embryo viability rates, questions have been raised by various countries’ regulatory agencies regarding the effects of LN2 exposure. The 0.25 ml plastic straw has long been used as an entirely closed vitrification system. However, the cooling rate is slower using the 0.25 ml straw than when using the ultra-rapid cooling method, resulting in injury to embryos exposed to a high concentration of cryoprotectant. Recently, a new system has been developed which addresses both the issues of rates of cooling and safeguards against LN2. This paper compares the clinical results of blastocyst vitrification and warming using the conventional 0.25 ml straw method with those of the Rapid-i™ method. The survival rate after warming the group cryopreserved using Rapid-i™ was significantly higher than that of the group of frozen-thawed cryopreserved using the 0.25 ml straw. Moreover, the on-going pregnancy rate after transfer blastocysts into recipients’ uteri was higher after cryopreservation with the Rapid-i™ method. These results suggest that the new closed system is safe to use in cryopreservation.

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