As several cases of chimerism have been reported in pregnancy by assisted reproductive technology (ART), ART-related problems include not only an increased risk of multiple pregnancy but also a possibility of chimerism. In particular, confined blood chimerism (CBC), which is attributable to placental vessel anastomosis between twins, has been detected in 11 cases of monochorionic dizygotic twins. It is possible that three or four alleles could be detected in patients with CBC, misleading physicians at the time of blood transfusion or genotyping for transplantation of an allograft. Therefore, sufficient informed consent and genetic counseling prior to ART are absolutely necessary for patient quality of life. ART may increase the chance of cell fusion (or cell cleavage) during embryogenesis, causing a increased risk of chimerism. Elective single-embryo transfer could avoid chimerism caused by cell-fusion after embryo transfer, but not chimerism generated before embryo transfer. To clarify the problem of chimerism in pregnancy by ART and determine solutions for this problem, both long-term follow-up of cases of chimerism and molecular genetic analysis using DNA polymorphic markers are essential.

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On the other side of the worldwide advance in assisted reproductive technology (ART), there is increasing concern about the maternal, fetal and post-natal risks of ART. To avoid multiple pregnancies, use of elective single embryo transfer (eSET) has been suggested in north European countries. In this review, we attempted to present an evidence-based discussion of the risks/benefits of ART and eSET. While many perinatal risks might be worse in ART than with natural conception in singleton pregnancies, the case is different in twin pregnancies and mainly depends on their monochorionic nature. Especially for early detection of intrauterine growth restriction, it is important to pay special attention to the location of umbilical cord insertion in the placenta. To evaluate the effectiveness of eSET, more studies are necessary with randomized controls.

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Extended embryo culture has brought many benefits to ART (assisted reproduction technologies). However, it is difficult to establish a culture system that mimics physiological conditions completely, and there is concern that artificial conditions could affect human development. Epigenetic modifications, such as DNA methylation, alter gene functions without mutations of gene sequences and are essential mechanisms for normal human development. For example, aberrant DNA methylation patterns (epimutations) actually cause some congenital anomaly syndromes. Since DNA methylation is a reversible chemical modification, it has been shown experimentally that epimutations can be introduced into embryos by environmental conditions. Several reports have suggested that ART may lead to epimutations in children and cause developmental defects. Detailed epidemic and biological studies of the relations among all these events are necessary.

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Human sperm has functional domains on the head and tail (flagellum). Since functional molecules are organized into the substructures of these domains, sperm with good appearance are expected to be fertile. Thus, it is of interest to see how sperm fine morphologies relate to fertility. The sperm head is divided into the acrosome and postacrosome regions. The acrosome region is further divided into the anterior acrosome for the acrosome reaction and posterior acrosome (equatorial segment) for gamete membrane fusion. The postacrosome region is involved in egg activation. In addition, the human sperm head has varieties of nuclear vacuoles. High-resolution light microscopy, which has recently been used in IMSI (intracytoplasmic morphologically selected sperm injection), is helpful in identifying vacuoles. However, IMSI is still insufficient for analysis of the contents of vacuoles; electron microscopy and evaluation tests for sperm DNA damage are more helpful for profound analysis of the nature of nuclear vacuoles and DNA status, respectively. The neck region carries the paternal centrosome to the oocyte to evoke sperm aster, from which microtubules emanate. Organization failure of the midpiece and principal piece strongly affects motility. This paper discusses the relationship beween the normality/abnormality of sperm substructures and fertility and shows typical phenotypes found in gene knockout animal models.

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The effect of IMSI on avoiding fertilization with chromosomally abnormal sperm was examined in the present study. Normal shaped human sperm with large vacuoles were identified at 1,000× magnification on an inverted microscope with a 100× oil objective lens and were then cytogenetically analyzed after injection into mouse oocytes. In all semen samples from ten consenting patients receiving IVF treatment, more than 90% of the oval shaped sperm were determined to be abnormal, having vacuoles of various sizes on the head. Our cytogenetic analysis showed that the incidence of chromosome aberrations did not differ in sperm with large vacuoles from infertile patients and normal sperm from fertile donors. In addition, the risk of structural chromosome aberrations deriving from DNA lesions was very low in sperm with large vacuoles (9.1%) compared with elongated sperm (33.3%), the data for which was previously reported by us (2004). In conclusion, sperm DNA fragmentation dose not seem to explain the phenomenon reported by Berkovitz et al. (2006) in which injection of sperm with large vacuoles reduced pregnancy outcome. We also doubt whether IMSI is needed, since sperm with large vacuoles were easily identified at even 400× magnification.

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In conventional IVF, a sperm penetrates an ovum through natural selection, and embryologists sort sperm for ICSI. The quality of the original ovum and sperm has the greatest effect on the quality of the fertilized egg (embryo). Therefore, the selection of superior sperm is critical for ICSI. Although sperm cells are 20-fold smaller than embryos or ova, they are observed and evaluated for ICSI using an inverted microscope at ×400 magnification usually used for observation of embryos or ova. Recently, a technique called intracytoplasmic morphologically selected sperm injection (IMSI) has attracted attention. This technique uses an inverted microscope with higher magnification and resolution than that used for ICSI, which enables embryologists to precisely observe sperm morphology, particularly the presence or absence of vacuoles in the sperm head, and to sort sperm for microfertilization. Although IMSI has some technological problems, including a narrow microscopic field due to higher magnification that leads to delicate microscopic operation, its clinical efficacy has been recognized. In the present review, we describe the status of IMSI outside Japan and the current practice of IMSI at Kiba Park Clinic.

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In ICSI, sperm is selected based on its motility and shape under 200-400× magnification. However, sperm morphology, especially the existence of vacuoles, may relate to sperm nuclear DNA fragmentation. Bartoov et al. reported the application of sperm selection under high (1,000-6,000×) magnification, which they referred to as IMSI (intracytoplasmic morphologically-selected sperm injection). In our initial trials, the fertilization, blastocyst development, pregnancy and miscarriage rates of 115 single blastocyst transfer cycles with IMSI or ICSI were 84.0% vs. 75.5%, 47.5% vs. 43.7%, 50.0% vs. 47.4% and 10.0% vs. 11.0%, respectively. Further studies are required to establish the systems and clarify the indications for IMSI.

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We observed the crater defects (CD) of human sperm head at a magnification of ×1,000 with an oil immersion lens and using an inverted microscope equipped with Nomarski optics and scanning and transmission electron microscope. Motile and normally shaped sperm with three different sizes of CD were selected and injected into M-II oocytes, donated by a consenting human patient and superovulated mice. On average, 1.54 CDs were confirmed in almost all (95.9%) sperm head, and the incidences of CDs did not change after the various demembraned pretreatments. The CDs were also found in epididymal and testicular sperm and elongating and elongated spermatids, but the incidences were a little fewer than ejaculated sperm. No CDs were found in mouse and boar sperm irrespective of the pretreatments. The fertilization and blastocyst formation rates were not significantly different following the ICSI using human sperm with 3 sizes of CDs. We speculate that CDs are not abnormal findings and physically structural changes during sperm maturation process.

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The growing oocytes that are present in bovine ovaries in large numbers can be a potential resource for future livestock production. Here we describe a culture system that allows growing oocytes to survive for an extremely long period. Preantral follicles or oocyte-granulosa cell complexes in early antral follicles were isolated from bovine ovaries. The complexes/follicles were classified into three groups according to oocyte size, 90.0-99.5 μm (Class I), 80.0-89.5 μm (Class II), 70.0-79.5 μm (Class III), and were cultured for 20, 28 and 36 days, respectively. The rate of oocyte recovery decreased as the culture period increased. During the culture period, the mean oocyte size in Class I increased to about 115 μm. The mean diameters of Class II and III at the end of the culture periods were 107 μm and 102 μm, respectively. Since many oocytes, especially in Class II and III, still needed to grow to achieve full size, extension of the culture period and/or substantial improvement of the culture conditions are likely required.

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Soybean products contain high concentrations of phytoestrogens. Isoflavone is the major phytoestrogen in soy and has been shown to display estrogen-like activities. It is well known that the delivery of isoflavone components across the placenta and in milk occurs in fetuses and infants following maternal exposure to isoflavone. We examined the effect of in utero and lactational exposure to isoflavone on the development and fertility of mouse offspring over two generations (F1 and F2). Pregnant mice were given daily subcutaneous injections of isoflavone (5, 50, 100 mg/kg/day) during the gestational and lactational stages until 20 days after delivery. At postnatal day (PND) 42, the F1 pups were sacrificed, and their body masses and organs (liver, kidney, testis, epididymis, ovary and uterus) were weighed. The other F1 pups were mated with non-treated mice or F1 heterosexual mice to estimate F1 fertility and development of F2 pups. The body weights of F1 pups increased at dose of 5 mg/kg/day. The epididymis weights of F1 males increased at doses of 5 and 100 mg/kg/day. No significant differences were found in the uterus and ovary weights of F1 females in all treatments. In the F2 males, the body weights of the 50 mg/kg/day group were higher than those of the control group. The epididymis weights of the 5 and 100 mg/kg/day groups were higher than those of the control group. In the F2 females, the body weights of the 5 and 100 mg/kg/day groups and the ovary weights of the 5 and 50 mg/kg/day groups were higher than those of the control group. There was no significant difference in pregnancy rate or numbers of live or dead fetuses in any treatment for the F1 and F2 offspring. These results suggest that gestational and lactational exposure to isoflavone presumably affect the body weights and some reproductive organ weights of F1 and F2 offspring, but are not seriously harmful to potential fertility.

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