The Natural History of Spontaneously Occurred Endometriosis in Cynomolgus Monkeys by Monthly Follow-Up Laparoscopy for Two Years.

Endometriosis, a disease in which endometrial tissue proliferates outside the uterus, is a progressive disease that affects women in reproductive age. It causes abdominal pain and infertility that severely affects the quality of life in young women. The mechanism of the onset and development of endometriosis has not been fully elucidated because of the complex mechanism involved in the disease. Nonhuman primates have been used to study the pathogenesis of spontaneous endometriosis because of their gynecological and anatomical similarities to humans. To reveal the natural history of endometriosis in cynomolgus monkeys, we selected 11 female cynomolgus monkeys with spontaneous endometriosis and performed monthly laparoscopies, mapping endometriotic lesions and adhesions up to two years. At the initial laparoscopy, endometriotic lesions were exclusively found in the vesicouterine pouch in 45.4% (5/11) of the monkeys and spread to the Douglas' pouch over time. Appearance of small de novo lesions and disappearance of some of the small lesions were observed in 100% (11/11) and 18.2% (2/11) of the monkeys, respectively. Endometriosis developed in all monkeys, and the speed of progression varied greatly among individuals that could be attributed to the degree or frequency of retrograde menstruation and genetic factors; these findings support the similarities between humans and monkeys, thus verifying the value of this nonhuman primate model. Finding reliable quantification markers and unravelling the underlying factors in correlation with the spatiotemporal development of the disease using a nonhuman primate model would be useful for the better management of endometriosis in humans.

Suppressive regulatory T cells and latent transforming growth factor-ß-expressing macrophages are altered in the peritoneal fluid of patients with endometriosis.

BACKGROUND: Endometriosis is a known cause of infertility. Differences in immune tolerance caused by regulatory T cells (Tregs) and transforming growth factor-ß (TGF-ß) are thought to be involved in the pathology of endometriosis. Evidence has indicated that Tregs can be separated into three functionally and phenotypically distinct subpopulations and that activated TGF-ß is released from latency-associated peptide (LAP) on the surfaces of specific cells. The aim of this study was to examine differences in Treg subpopulations and LAP in the peripheral blood (PB) and peritoneal fluid (PF) of patients with and without endometriosis. METHODS: PB and PF were collected from 28 women with laparoscopically and histopathologically diagnosed endometriosis and 20 disease-free women who were subjected to laparoscopic surgery. Three subpopulations of CD4+ T lymphocytes (CD45RA+FoxP3low resting Tregs, CD45RA-FoxP3high effector Tregs, and CD45RA-FoxP3low non-Tregs) and CD11b+ mononuclear cells expressing LAP were analyzed by flow cytometry using specific monoclonal antibodies. RESULTS: Proportions of suppressive Tregs (resting and effector Tregs) were significantly higher in the PF samples of patients with endometriosis than in those of control women (P = 0.02 and P < 0.01, respectively) but did not differ between the PB samples of patients and controls. The percentage of CD11b+LAP+ macrophages was significantly lower in PF samples of patients with endometriosis than in those of controls (P < 0.01) but was not altered in the PB samples. CONCLUSION: Proportions of suppressive Tregs and LAP+ macrophages are altered locally in the PF of endometriosis patients.

Salvage of fetal karyotype information from SNP array data obtained from products of conception with maternal cell contamination.

OBJECTIVE: Maternal cell contamination (MCC) is known to increase the risk of misdiagnosis in prenatal diagnosis as well as in diagnostic tests for the products of conception (POC) from miscarriages. We aimed to develop a data correction method to salvage fetal karyotype information from single-nucleotide polymorphism (SNP) array data for POC with MCC when parental genotype data are available. METHODS: We obtained SNP array data from mixed genomic DNAs of a mother-child pair and used the dataset to assess the accuracy of data correction. We subsequently applied our method to miscarriage specimens with MCC. RESULTS: We adopted a linear interpolation model as a data correction method and implemented the method in an R package, snpsal. We successfully determined the fetal karyotypes of two miscarriage specimens that were previously undiagnosed due to MCC to be normal in one case and trisomy 16 in the other case using snpsal. CONCLUSION: The R package, snpsal, developed in this study facilitates rapid and accurate estimation of the fetal karyotype from SNP array data for POC with MCC. © 2017 John Wiley & Sons, Ltd.

Exacerbation of Endometriosis Due To Regulatory T-Cell Dysfunction.

Context: Endometriosis is a chronic inflammatory disease associated with altered immune response to endometrial cells facilitating the implantation and proliferation of ectopic endometrial tissues. Although regulatory T (Treg) cells play a key role in T cell-mediated immune response and development of immune disorders, their significance in endometriosis remains to be elucidated. Recently, CD4+CD45RA- forkhead box protein 3 (Foxp3)hi T cells, activated Treg cells, have been identified as a functionally true suppressive population of Treg cells. Objective: To investigate the role of Treg cells in endometriosis. Design: Three Treg cell fractions (resting Treg cells, activated Treg cells, and non-Treg cells) were examined using flow cytometry in the endometrioma, endometrium, peritoneal fluid, and peripheral blood obtained from women with (n = 27) and without (n = 28) endometriosis. A mouse model of endometriosis was made in Foxp3tm3Ayr/J (Foxp3DTR) C57BL/6 Treg cell-depleted mice (n = 28). Results: In women with endometrioma, the proportion of activated Treg cells in the endometrioma and the endometrium, but not in the peritoneal fluid or peripheral blood, was significantly decreased compared with that in women without endometriosis. In Foxp3DTR/diphtheria toxin mice, the number and weight of endometriotic lesions, inflammatory cytokine levels and angiogenetic factors were significantly increased compared with those in control mice. Conclusions: Treg cell deficiency exaggerates local inflammation and angiogenesis and simultaneously facilitates the attachment and growth of endometrial implants. The findings provide an insight into dysregulated immune response for the pathogenesis and development.

Dual Positive Regulation of Embryo Implantation by Endocrine and Immune Systems--Step-by-Step Maternal Recognition of the Developing Embryo.

In humans, HCG secreted from the implanting embryo stimulates progesterone production of the corpus luteum to maintain embryo implantation. Along with this endocrine system, current evidence suggests that the maternal immune system positively contributes to the embryo implantation. In mice, immune cells that have been sensitized with seminal fluid and then the developing embryo induce endometrial differentiation and promote embryo implantation. After hatching, HCG activates regulatory T and B cells through LH/HCG receptors and then stimulates uterine NK cells and monocytes through sugar chain receptors, to promote and maintain pregnancy. In accordance with the above, the intrauterine administration of HCG-treated PBMC was demonstrated to improve implantation rates in women with repeated implantation failures. These findings suggest that the maternal immune system undergoes functional changes by recognizing the developing embryos in a stepwise manner even from a pre-fertilization stage and facilitates embryo implantation in cooperation with the endocrine system.

Robust In Vitro Induction of Human Germ Cell Fate from Pluripotent Stem Cells.

Mechanisms underlying human germ cell development are unclear, partly due to difficulties in studying human embryos and lack of suitable experimental systems. Here, we show that human induced pluripotent stem cells (hiPSCs) differentiate into incipient mesoderm-like cells (iMeLCs), which robustly generate human primordial germ cell-like cells (hPGCLCs) that can be purified using the surface markers EpCAM and INTEGRINα6. The transcriptomes of hPGCLCs and primordial germ cells (PGCs) isolated from non-human primates are similar, and although specification of hPGCLCs and mouse PGCs rely on similar signaling pathways, hPGCLC specification transcriptionally activates germline fate without transiently inducing eminent somatic programs. This includes genes important for naive pluripotency and repression of key epigenetic modifiers, concomitant with epigenetic reprogramming. Accordingly, BLIMP1, which represses somatic programs in mice, activates and stabilizes a germline transcriptional circuit and represses a default neuronal differentiation program. Together, these findings provide a foundation for understanding and reconstituting human germ cell development in vitro.

Cutaneous exposure to agglomerates of silica nanoparticles and allergen results in IgE-biased immune response and increased sensitivity to anaphylaxis in mice.

BACKGROUND: The skin is a key route of human exposure to nanomaterials, which typically occurs simultaneously with exposure to other chemical and environmental allergen. However, little is known about the hazards of nanomaterial exposure via the skin, particularly when accompanied by exposure to other substances. RESULTS: Repeated topical treatment of both ears and the shaved upper back of NC/Nga mice, which are models for human atopic dermatitis (AD), with a mixture of mite extract and silica nanoparticles induced AD-like skin lesions. Measurements of ear thickness and histologic analyses revealed that cutaneous exposure to silica nanoparticles did not aggravate AD-like skin lesions. Instead, concurrent cutaneous exposure to mite allergens and silica nanoparticles resulted in the low-level production of allergen-specific IgGs, including both the Th2-related IgG1 and Th1-related IgG2a subtypes, with few changes in allergen-specific IgE concentrations and in Th1 and Th2 immune responses. In addition, these changes in immune responses increased the sensitivity to anaphylaxis. Low-level IgG production was induced when the mice were exposed to allergen-silica nanoparticle agglomerates but not when the mice exposed to nanoparticles applied separately from the allergen or to well-dispersed nanoparticles. CONCLUSIONS: Our data suggest that silica nanoparticles themselves do not directly affect the allergen-specific immune response after concurrent topical application of nanoparticles and allergen. However, when present in allergen-adsorbed agglomerates, silica nanoparticles led to a low IgG/IgE ratio, a key risk factor of human atopic allergies. We suggest that minimizing interactions between nanomaterials and allergens will increase the safety of nanomaterials applied to skin.

Intestinal absorption and biological effects of orally administered amorphous silica particles.

Although amorphous silica nanoparticles are widely used in the production of food products (e.g., as anticaking agents), there is little information available about their absorption and biological effects after oral exposure. Here, we examined the in vitro intestinal absorption and in vivo biological effects in mice of orally administered amorphous silica particles with diameters of 70, 300, and 1,000 nm (nSP70, mSP300, and mSP1000, respectively) and of nSP70 that had been surface-modified with carboxyl or amine groups (nSP70-C and nSP70-N, respectively). Analysis of intestinal absorption by means of the everted gut sac method combined with an inductively coupled plasma optical emission spectrometer showed that the intestinal absorption of nSP70-C was significantly greater than that of nSP70. The absorption of nSP70-N tended to be greater than that of nSP70; however, the results were not statistically significant. Our results indicate that silica nanoparticles can be absorbed through the intestine and that particle diameter and surface properties are major determinants of the degree of absorption. We also examined the biological effects of the silica particles after 28-day oral exposure in mice. Hematological, histopathological, and biochemical analyses showed no significant differences between control mice and mice treated with the silica particles, suggesting that the silica nanoparticles evaluated in this study are safe for use in food production.

Induction of pluripotent stem cells from fetal and adult cynomolgus monkey fibroblasts using four human transcription factors.

Induced pluripotent stem (iPS) cells have the potential to become a universal resource for cell-based therapies in regenerative medicine; however, prior to the use of such iPS cell-based therapies, preclinical assessment of their safety and efficacy is essential. Non-human primates serve as valuable animal models for human diseases or biomedical research; therefore, in this study, we generated cynomolgus monkey iPS cells from adult skin and fetal fibroblast cells by the retrovirally mediated introduction of four human transcription factors: c-Myc, Klf4, Oct3/4, and Sox2 (the so-called "Yamanaka factors"). Twenty to 30 days after the introduction of these factors, several cynomolgus monkey embryonic stem (ES) cell-like colonies appeared on SNL and mouse embryonic fibroblast (MEF) feeder layers. These colonies were picked and cultivated in primate ES medium. Seven iPS cell lines were established, and we detected the expression of pluripotent markers that are also expressed in ES cells. Reverse transcription polymerase chain reaction (PCR) showed that these iPS cells expressed endogenous c-Myc, Klf4, Oct3/4, and Sox2 genes, whereas several transgenes were silenced. Embryoid body and teratoma formation showed that the cynomolgus iPS cells had the developmental potential to differentiate into cells of all three primary germ layers. In summary, we generated cynomolgus monkey iPS cells by retrovirus-mediated transduction of the human transcription factors, c-Myc, Klf4, Oct3/4, and Sox2 into adult cynomolgus monkey skin cells and fetal fibroblasts. The cynomolgus monkey is the most relevant primate model for human disease, and the highly efficient generation of monkey iPS cells would allow investigation of the treatments of various diseases in this model via therapeutic cloning.

Prevention of premature ovarian failure and osteoporosis induced by irradiation using allogeneic ovarian/bone marrow transplantation.

BACKGROUND: Two side effects of irradiation are premature ovarian failure (POF) and osteoporosis, both of which are concerns not only clinically, for patients, but also experimentally, for animals. We examine whether bone marrow transplantation (BMT) can correct the POF induced by radiation and also address whether allogeneic ovarian transplantation (OT) can modulate the adverse effects of radiotherapy. METHODS: Eight-week-old female C57BL/6 mice were lethally irradiated with 6 Gy x2, and then injected with allogeneic bone marrow cells into their bone marrow cavity using our previously described intrabone marrow (IBM)-BMT technique. Allogeneic ovaries were simultaneously transplanted under the renal capsules of the mice. RESULTS: Three months after the transplantation, we noted that hematopoietic and lymphoid cells had been successfully reconstituted. The ovaries transplanted under the renal capsules demonstrated signs of development with a large number of differentiating follicles at different stages of development. Importantly, the total bone mineral density of the tibia in the "IBM-BMT+OT" (BMT/OT) group remained normal. However, the reproductive function of the recipient mice was not restored, despite the presence of many immature oocytes in the host ovaries in the BMT/OT group. In the BMT group, no oocytes were found in the host ovaries. CONCLUSIONS: These findings suggest that IBM-BMT with ovarian allografts can be advantageous for young women with POF and osteopenia or osteoporosis that is due to chemotherapy and radiotherapy for malignant diseases.

Gender ratio distortion in abortuses and live births from patients with recurrent spontaneous abortion.

PROBLEM: Gender ratio of live birth in humans is approximately 1.05 and males are born a slightly more, while gender ratio of fertilization should be 1.00, suggesting that female fetus might be more sensitive to abortion than male fetus during pregnancy. METHOD OF STUDY: We examined karyotype of abortuses from patients with recurrent spontaneous abortion (RSA), who had at least one live birth before or after the treatment of RSA. RESULTS: Chromosomal abnormality was not frequent (14.6%) in the abortuses from the RSA patients. Among abortuses without chromosomal abnormality, male karyotype was rare (9.2%), and this gender ratio distortion was more prominent in RSA cases not carrying autoantibodies (3.5%) than that in the RSA cases carrying autoantibodies (26.3%), with statistical significance (P = 0.009). CONCLUSION: These observations suggested that the aborted fetuses from RSA of unknown etiology, i.e. no chromosomal abnormality and no autoantibody, were preferentially female.

Morphogenesis of polycystic ovaries as assessed by pituitary-ovarian androgenic function.

Despite polycystic ovaries (PCO) being a common morphology in women with polycystic ovary syndrome and regular menstruation, the regulatory principles in the morphogenesis of antral follicles have not yet been elucidated. In recognition of the complementary interaction between androgen-induced expression of the FSH receptor and FSH-augmented expression of the androgen receptor in granulose cells of antral follicles, a possible correlation of antral follicle count (AFC) and pituitary-ovarian androgenic function was investigated in 180 infertile women over days 3-5 of the menstrual cycle. Six discrete types of PCO with decreasing pituitary-ovarian androgenic function were identified: Type I (classical Stein-Leventhal syndrome), Type II (hyperandrogenemism), Type III (singular hyper-LH), Type IV (cryptic hyperandrogenism), Type V (relative LH dominancy) and Type VI (relative FSH dominancy), in parallel to a diminishing number of AFC from Type I to Type VI. Because during the early follicular phase of the cycle until the selection of the dominant follicle, antral follicles are composed of newly emerged healthy follicles plus atretic antral follicles that remain non-ovulated from previous cycles, it is proposed that the six types of PCO may represent the folliculogenetic spectra along which PCO morphogenesis proceeds.

Hyaluronan fragments generated by sperm-secreted hyaluronidase stimulate cytokine/chemokine production via the TLR2 and TLR4 pathway in cumulus cells of ovulated COCs, which may enhance fertilization.

The toll-like receptor (TLR) system is expressed in cumulus cells of ovulated cumulus-oocyte complexes (COCs) and is activated by bacterial lipopolysaccharides (LPS). However, the endogenous ligand(s) for the TLRs and the physiological role(s) in ovulated COCs remain to be defined. Based on reports that hyaluronan fragments can activate TLR2 and TLR4 in macrophages, and that ovulated COCs are characterized by a hyaluronan-rich matrix, we cultured ovulated mouse COCs with purified hyaluronan fragments, treated them with purified hyaluronidase or exposed them to sperm as a physiologically relevant source of hyaluronidase. Hyaluronan fragments or hyaluronidase activated the NFkappaB pathway and induced Il6, Ccl4 and Ccl5 mRNA expression within 2 hours. Anti-TLR2 and anti-TLR4 neutralizing antibodies significantly suppressed hyaluronan fragment- and hyaluronidase-induced activation of the NFkappaB pathway and the expression of these genes. When ovulated COCs were cultured with sperm, the expression and secretion of cytokine/chemokine family members were induced in a time-dependent manner that could be blocked by TLR2/TLR4 antibodies or by a hyaluronan-blocking peptide (Pep-1). The chemokines secreted from TLR2/TLR4-stimulated COCs activated cognate chemokine receptors (CCRs) localized on sperm and induced sperm protein tyrosine phosphorylation, which was used as an index of capacitation. Significantly, in vitro fertilization of COC-enclosed oocytes was reduced by the TLR2/TLR4 neutralizing antibodies or by Pep-1. From these results, we propose that TLR2 and TLR4 present on cumulus cells were activated by the co-culture with sperm in a hyaluronan fragment-dependent manner, and that chemokines secreted from COCs induced sperm capacitation and enhanced fertilization, providing evidence for a regulatory loop between sperm and COCs during fertilization.

Prevention of osteoporosis and hypogonadism by allogeneic ovarian transplantation in conjunction with intra-bone marrow-bone marrow transplantation.

BACKGROUND: We investigated the effects of ovarian allograft in conjunction with intra-bone marrow-bone marrow transplantation (IBM-BMT) on estrogen deficiency in mice. METHODS: Female C57BL/6 mice underwent ovariectomy (OvX). After 3 months, the mice were irradiated at 9.5 Gy, and the bone marrow cells (BMCs) of female BALB/c mice (8 weeks old) were then injected into the bone cavity of the B6 mice. Simultaneously, allogeneic ovaries from BALB/c mice were transplanted under the renal capsules of the B6 mice. RESULTS: Three months after the transplantation, the hematolymphoid cells were found to be completely reconstituted with donor-derived cells. The transplanted ovary tissues under the renal capsules were accepted without using immunosuppressants; there were a large number of growing follicles at different stages of development. Atrophic endometrium and its glands were also recovered by ovarian transplantation (OT). The transplanted allogeneic ovaries secreted estrogen at normal levels. Furthermore, bone loss was prevented to a certain extent. CONCLUSIONS: These findings suggest that IBM-BMT+OT will become a valuable strategy for young women with malignant tumors to prevent premature senescence, including hypogonadism and osteoporosis, after radiochemotherapy.

Ovarian theca cells in follicular function.

The role of theca cells in every aspect of ovarian follicular function is reviewed. A distinguishing feature of theca cells may be their ability to initiate follicle growth on differentiation from cortical stromal cells, stimulate follicle growth by granulosa cell mitosis through FSH-induced androgen receptor, and cause androgen-stimulated receptor formation of FSH. As LH not only stimulates androgen production by theca cells at tonic levels, but also induces morphological luteinization in addition to androgenesis at surge levels, the dual action concept of LH is proposed. Maturation of the selected dominant follicle and atresia of subordinate antral follicles is interpreted by this concept. Two-way signalling between oocytes and somatic theca cells with growth factors is shown to play a pivotal role in preantral folliculogenesis and atresia. Thus, theca cells have a more significant role in follicular function than previously thought.

Human leukocyte antigen may predict outcome of primary recurrent spontaneous abortion treated with paternal lymphocyte alloimmunization therapy.

PROBLEM: Recurrent spontaneous abortion (RSA) is defined by at least three consecutive abortions in otherwise healthy couples. Paternal lymphocyte alloimmunization therapy (PLAT) is an effective therapy for RSA in some cases, but there are no predictive markers about the effectiveness of PLAT. METHOD OF STUDY: Forty-two consecutive cases with primary RSA treated by PLAT and 23 controls were the subjects. Polymorphisms of human leukocyte antigen (HLA)-E, HLA-G, HLA-A, HLA-B, HLA-C and HLA-DRB1 were investigated by sequenced based typing. Promoter polymorphism and a 14 bp ins/del polymorphism in exon 8 were also investigated for HLA-G. RESULTS: Thirty-eight RSA wives became pregnant within 1 year after PLAT. Among them, 27 obtained babies (succeeded PLAT cases), while 11 again aborted with no detectable chromosomal abnormalities in the aborted fetuses (aborted PLAT cases). The frequencies of HLA-G*010401, A*2402, B*5201, and DRB1*1502 were significantly increased in the aborted cases than those in the succeeded cases or controls. Of note, HLA-G*010401 was found in all aborted cases whereas it was found in 51.9% of succeeded cases (odds ratio = 21.4, P = 0.006, P(c) = 0.03), and the presence of HLA-G*010401 could predict the abortion after PLAT with sensitivity and specificity of 100% and 48.1%, respectively. CONCLUSION: Human leukocyte antigen testing may be useful for predicting effectiveness of PLAT in RSA.

Assessment of human oocyte developmental competence by cumulus cell morphology and circulating hormone profile.

The predictive value of the morphology of the cumulus--oocyte complex (COC) has not yet been explored as a possible factor contributing to the success of human in-vitro maturation (IVM). In the present study, development-supporting competency of oocytes encircled in a large ( > or = 5) (grade A), moderate (3 approximately 4) (grade B) or small ( < or = 2) (grade C) number of cumulus cell layers was assessed, together with changes in hormonal profile following a truncated course of 150 IU pure FSH administration for 3 days prior to aspiration on laparoscopy indicated for endometriosis. FSH priming increased the number of COC aspirated without changing the proportion of the three morphological types of COC, which were then subjected to IVM in the presence of 200 mIU/ml FSH plus 1000 mIU/ml human chorionic gonadotrophin, followed by intracytoplasmic sperm injection. The highest development-supporting competence was observed not with oocytes in grade A COC harvested from natural cycles, but with oocytes in grade B COC from FSH-primed cycles. Hormonal profiles in patients bearing grade B COC were characterized by moderate response in oestradiol and progesterone production following FSH, with LH/FSH ratio being below 1.0. It is concluded that an optimal window of hormonal profile(s) may exist for follicle aspiration to obtain grade B COC in FSH-stimulated human IVM cycles.

Developmentally retarded frozen blastocysts can be rescued by synchronizing culture prior to transfer.

Asynchrony between embryo development and endometrial differentiation is the limiting step of successful pregnancy in assisted reproduction. The aim of this study was to investigate whether or not post-thaw synchronization culture of day 5-6 frozen embryos, prior to transfer, with endometrial differentiation resulted in pregnancy. A total of 142 cycles of 134 patients were transferred in three protocols. Blastocysts with cavities larger than half of the entire blastocyst volume were transferred without synchronizing culture on day 5 or 6 of progesterone commencement (P5/6) in hormone replacement treatment cycles (protocol 1). Blastocysts with cavitation below half of the entire blastocyst were cultured for 1 or 2 days after thawing prior to transfer on P5 or P6 (protocol 2). Morulae and very early stage blastocysts were thawed on the days corresponding to P5 and P6, and only the embryos that reached expanded or hatching blastocysts were transferred on P7 without synchronizing culture (protocol 3). Pregnancy rate in protocol 2 (32.0%) was comparable with that of protocol 1 (35.0%). It is concluded that developmentally retarded frozen embryos can be rescued with synchronizing culture prior to transfer by evading asynchrony.

Sperm-zona pellucida interaction and immunological infertility.

Immune reactions against gametes appear to be physiologically important for the maintenance of homeostasis in reproduction. In contrast, aberration of the immune homeostasis might give rise to 'immunological infertility'. Antisperm antibodies cause infertility by blocking fertilization. The mechanism can be explained as inhibiting the acrosome reaction of sperm by their blocking effect on capacitation through inhibiting an increase of fluidity of the sperm membrane. Autoantibodies against zona pellucida also cause infertility by blocking sperm-zona pellucida interaction, though the definitive mechanism has not been elucidated. Pretreatment of spermatozoa with D-mannnose completely inhibited sperm penetration through, but not binding to, the zona pellucida. Furthermore, very rapid kinetics between sperm extracts and D-mannnose by a BIAcore apparatus suggest that a D-mannose ligand of the sperm surface is easy to bind to and dissociate from a D-mannose residue in the sperm receptor site on the zona pellucida. Thus, D-mannnose on the human zona pellucida might be an essential molecule acting as a second sperm receptor, through which sperm penetrate into the zona pellucida. Because these antibodies appear to not cause any deleterious clinical symptoms, sperm and zona pellucida antigens are promising candidates in the development of an immunocontraceptive. (Reprod Med Biol 2006; 5: 95-104).

Analyses of peripheral blood mononuclear cells in operational tolerance after pediatric living donor liver transplantation.

Operational tolerance (graft acceptance in an immunosuppression (IS)-free environment) after living-donor liver transplantation (LDLT) could occur by our elective protocol in some patients. There is, nevertheless, no reliable parameter to monitor patients who may discontinue IS without a risk of rejection. To identify such parameters, we systemically phenotyped peripheral blood mononuclear cells from operationally tolerant patients. An increase was observed in the frequency of CD4+CD25high+ cells, B cells and Vdelta1/Vdelta2 gammadeltaT-cells ratio in operationally tolerant patients (Gr-tol; n = 12), compared with those from age-matched volunteers (Gr-vol; n = 24) or patients on IS (Gr-IS; n = 19). The frequency of NK cells was decreased in Gr-tol, compared with those in Gr-IS or Gr-vol. The frequency of NKT cells was decreased after LDLT, compared with that in Gr-vol. Although the contribution of those subsets to the tolerant state remains elusive, the results may provide important clues for reliable indicators of tolerance after LDLT.