The developmental potential of mature oocytes derived from rescue in vitro maturation.

OBJECTIVE: To examine the developmental competence of immature oocytes in stimulated cycles, that matured after rescue in vitro maturation (IVM) compared with their sibling in vivo matured oocytes. DESIGN: Retrospective cohort study. SETTING: IVF clinic. PATIENTS: A total of 182 patients underwent 200 controlled ovarian stimulation cycles with intracytoplasmic sperm injection cycles in which immature oocytes were retrieved and at least one mature oocyte was obtained through rescue IVM. INTERVENTION: In vitro culture of immature germinal vesicle (GV) and metaphase I (MI) oocytes, retrieved in stimulated cycles. MAIN OUTCOME MEASURES: Fertilization rate, cleavage rate, blastulation rate, ploidy of embryos evaluated using preimplantation genetic testing for aneuploidy, morphokinetic parameters and pregnancy outcomes. RESULTS: In total, 2,288 oocytes were retrieved from 200 cycles. After denudation, 1,056 of the oocytes (46% ± 16%) were classified as metaphase II (MII). A total of 333/375 (89%) of MI oocytes and 292/540 (54%) of GV oocytes matured overnight and underwent intracytoplasmic sperm injection. The fertilization rates of matured oocytes from MI rescue IVM (R-MI) and from GV rescue IVM (R-GV) were comparable with those of their sibling MII oocytes (71% vs. 66%; 66% vs. 63%, respectively). Early cleavage rates (80% ± 35% vs. 92% ± 20%; 80% ± 42% vs. 95% ± 28%, respectively) and blastulation rates (32 ± 40% vs. 62 ± 33%; 24 ± 37% vs. 60 ± 35%, respectively) were significantly decreased in rescue IVM matured oocytes (R-oocytes)-derived zygotes, but the blastocyst (BL) euploidy rate and "good quality" BL rate were comparable with those of MII sibling-derived embryos. In addition, rescue IVM embryos showed significantly higher levels of multinucleation at the 2- and 4-cell stages, as well as higher rates of zygote direct cleavage from one to 3 to 4 cells. Overall, 21 transfers of rescue IVM embryos resulted in 3 healthy live births. CONCLUSIONS: For patients with a low maturation rate and/or low numbers of mature oocytes at retrieval, rescue IVM may contribute more competent oocytes and additional viable BLs for transfer from the same stimulation cycle, maximizing the chances for pregnancy and live birth.

Sperm content of postacrosomal WW binding protein is related to fertilization outcomes in patients undergoing assisted reproductive technology.

OBJECTIVE: To determine the levels of postacrosomal WW binding protein (PAWP) in the spermatozoa of men that were used clinically for intracytoplasmic sperm injection (ICSI) and to correlate them with infertility treatment outcomes. DESIGN: Prospective clinical and laboratory study. SETTING: University-based laboratory and infertility clinic. PATIENT(S): Men undergoing ICSI for the treatment of couples' infertility (n=110). INTERVENTION(S): Quantitative analysis of sperm PAWP levels by flow cytometry and developmental analysis of PAWP expression by immunoblotting, immunofluorescence, and immunohistochemistry. MAIN OUTCOME MEASURE(S): PAWP flow-cytometric levels and immunolocalization in spermatozoa. RESULT(S): A strong positive correlation was found between PAWP expression levels and fertilization rates after ICSI, with high levels of PAWP being associated with higher fertilization rates; the positive correlation was independent of age, DNA fragmentation index, and other sperm parameters. PAWP expression levels were correlated with embryonic development, with high levels of PAWP being associated with a lower number of arrested embryos within 3-5 days post-ICSI. PAWP expression was detected during the late stages of human spermiogenesis in elongating spermatids, confirming previous findings in various animal models. CONCLUSION(S): Our clinical data from infertile couples demonstrate significant correlations between sperm PAWP levels and both fertilization rates and normal embryonic development after ICSI. Considering its proposed role in the initiation of oocyte activation, we suggest that PAWP could have potential applications in the diagnosis and treatment of infertility.

Sperm-derived WW domain-binding protein, PAWP, elicits calcium oscillations and oocyte activation in humans and mice.

Mammalian zygotic development is initiated by sperm-mediated intracellular calcium oscillations, followed by activation of metaphase II-arrested oocytes. Sperm postacrosomal WW binding protein (PAWP) fulfils the criteria set for an oocyte-activating factor by inducing oocyte activation and being stored in the perinuclear theca, the sperm compartment whose content is first released into oocyte cytoplasm during fertilization. However, proof that PAWP initiates mammalian zygotic development relies on demonstration that it acts upstream of oocyte calcium oscillations. Here, we show that PAWP triggers calcium oscillations and pronuclear formation in human and mouse oocytes similar to what is observed during intracytoplasmic sperm injection (ICSI). Most important, sperm-induced calcium oscillations are blocked by coinjection of a competitive inhibitor, derived from the WWI domain-binding motif of PAWP, implying the requirement of sperm PAWP and an oocyte-derived WWI domain protein substrate of PAWP for successful fertilization. Sperm-delivered PAWP is, therefore, a unique protein with a nonredundant role during human and mouse fertilization, required to trigger zygotic development. Presented data confirm our previous findings in nonmammalian models and suggest potential applications of PAWP in the diagnosis and treatment of infertility.-

Is the zona pellucida thickness of human embryos influenced by women's age and hormonal levels?

OBJECTIVE: To evaluate whether zona pellucida thickness (ZPT) of human embryos is correlated with maternal age, patient's hormonal status, embryo quality, and IVF outcomes. DESIGN: Prospective study. SETTING: University-affiliated IVF clinic. PATIENT(S): Couples undergoing IVF-ET cycles. INTERVENTION(S): Zona measurements, clinical data collection. MAIN OUTCOME MEASURE(S): Correlation between the ZPT and maternal age, basal FSH and E(2) levels, stimulation protocols, cause of infertility, embryo quality, and implantation/pregnancy rates. RESULT(S): The measurements of ZPT were collected from 5,184 day 3 human embryos originated from 744 IVF patients. The overall mean ZPT was 16.18 ± 2.00 µm. No significant correlation was observed between the ZPT and the patient's age, E(2) values on the day of hCG administration, basal concentration of serum FSH, stimulation protocol, infertility diagnosis, and implantation/pregnancy rates. The ZPT was strongly influenced only by the embryo quality: Embryos with good morphology exhibited considerably thinner ZP compared with those of less favorable morphology (mean 15.87 ± 2.48 µm vs. 16.36 ± 2.57 µm, respectively). The ZPT had no significant impact on the implantation and pregnancy rates. CONCLUSION(S): The thickness of the human ZP of day 3 embryos is not influenced by women's age and hormonal levels. The strong correlation between ZPT and embryo quality suggests that thickness of ZP depends on inherent embryo properties. The overall ZPT is not a good predictive indicator for IVF clinical outcomes.

Trafficking of peripheral blood CD56(bright) cells to the decidualizing uterus--new tricks for old dogmas?

CD56(bright) lymphocytes become abundant in the human uterus during every menstrual cycle, following the surge in pituitary-derived luteinizing hormone (LH), which initiates final oocyte maturation. While the uterus is host to some CD56(bright) cells prior to ovulation, the rapid increase is thought to be due to proliferation of the resident population, accompanied by recruitment of CD56(bright) lymphocytes from the circulation. The rapid increase in CD56(bright) cells is concurrent with the onset of decidualization, the transformation of uterine stromal cells into secretory decidual cells. Uterine CD56(bright) cells proliferate and differentiate to become the predominant lymphocytes of the post-ovulatory uterus. These distinct, tissue-specific natural killer (NK) cells either die prior to menses or increase in number during early pregnancy, and then decline toward the end of the first trimester. Since lymphocytes home to tissues from the circulation, we investigated mechanisms of NK cell traffic over the course of natural menstrual cycles by measuring functional interactions between CD56+ cells from blood and endothelial cells using the Stamper-Woodruff assay of lymphocyte adhesion to frozen tissue sections. While a baseline level of adhesion was maintained throughout the cycle, elevated l-selectin-dependent adhesion of peripheral blood CD56(bright) cells occurred during a peri-ovulatory window. However, there were no significant menstrual cycle-induced changes in the transcription of l-selectin, alpha 4 integrin or LFA-1, or in expression of these proteins by NK cells, suggesting that the enhanced adhesion was due to post-translational modifications of these molecules. Quantitative RT-PCR failed to amplify the message for LH receptor or the alpha or beta forms of progesterone or estrogen receptors from blood NK cell subsets. Thus, we conclude that the actions of LH, E(2,) and P(4) on NK cells that promote interactions with endothelium and potential uterine homing are indirectly mediated through the responsiveness of other cell types.

Periovulatory increases in tissue homing potential of circulating CD56(bright) cells are associated with fertile menstrual cycles.

CD56(bright) lymphocytes appear in the uterus 3-5 d after ovulation coincident with the onset of stromal cell decidualization. Although the source of these uterine immune cells is not defined, a subset of blood CD56(bright) cells exhibits enhanced capacity to adhere to decidual vascular endothelium during the periovulatory period of menstrual cycles. In this study, the effects of early pregnancy on the adhesive capacity of CD56(bright) cells to bind uterine substrates were examined in a time-course study of 18 infertile women undergoing natural cycles before transfer of frozen/thawed embryos and 18 infertile women undergoing controlled ovarian stimulation. There were three pregnancies in the natural cycle group and seven in the hormone-stimulated cohort. Hormone levels, and number and quality of transferred embryos were similar between pregnant and nonpregnant cycles. However, the adhesive function of CD56(bright) cells increased before ovulation in hormone-treated women who became pregnant and before embryo transfer in naturally cycling women who became pregnant. This pattern of incremental adhesion, which was less frequently observed in unsuccessful cycles, suggests a role for NK cells in implantation. These results support the idea that temporal control of NK cell homing to the uterine microenvironment is a prerequisite to pregnancy.

Menstrual cycle hormones induce changes in functional interactions between lymphocytes and decidual vascular endothelial cells.

During the secretory phase of the menstrual cycle, a natural killer (NK) cell subset expressing cluster of differentiation (CD)56bright appears in the decidualizing uterus and remains until onset of menses. If pregnancy occurs, decidual NK cells increase to become the predominant uterine lymphocytes of early pregnancy. To elucidate mechanisms of CD56bright cell recruitment to the uterus, an in vitro adhesion assay was used to assess the effect of the menstrual cycle, as well as cycle-associated hormones on adhesive properties of human lymphocytes. Adhesion of human peripheral blood lymphocytes to pregnant mouse lymph nodes and Peyer's Patches tissue sections was constant throughout the cycle. When uterine tissue was used as the substrate, adhesive CD56+ cells were found only in decidua basalis. Adhesion increased at the LH surge. Adhesion was mediated through both L-selectin and alpha4-integrin-dependent mechanisms. Furthermore, we observed increased adhesive function in CD56+ cells from male donors which had been cultured with estradiol or LH compared with cell aliquots cultured without additives. Lymphocytes adherent to mouse uterine tissue were predominantly CD56bright, suggesting that peripheral NK cells may be actively recruited to the uterus in an important, brief endocrine-regulated fashion at the time of ovulation to establish the decidual NK population of early pregnancy.

Coordinate regulation of lymphocyte-endothelial interactions by pregnancy-associated hormones.

Precursors of uterine NK cells home to the uterus during early pregnancy from multiple lymphohemopoietic sources. In mouse uterine tissue, pregnancy markedly up-regulates both L-selectin- and alpha(4) integrin-dependent adhesion pathways for circulating human CD56(bright) cells, the phenotype of human uterine NK cells. Based on roles for these adhesion molecules in lymphocyte homing, we examined effects of pregnancy or the steroid hormones 17beta-estradiol or progesterone on lymphocyte-endothelial interactions in secondary lymphoid tissues and in uterus. From preimplantation gestation day 3, specialized high endothelial venules in peripheral lymph nodes and Peyer's patches supported elevated L-selectin and alpha(4)beta(7) integrin-dependent lymphocyte adhesion under shear throughout pregnancy, as compared with high endothelial venules of virgin or postpartum donors. Squamous endothelium from nonlymphoid tissue was not affected. Pregnancy-equivalent endothelial responses were observed in lymph nodes and Peyer's patches from ovariectomized mice receiving 17beta-estradiol and/or progesterone replacement therapy. Adhesion of human CD56(bright) cells to uteri from pregnant or hormone-treated ovariectomized mice was enhanced through L-selectin- and alpha(4) integrin-dependent mechanisms and involved multiple vascular adhesion molecules including mucosal addressin cell adhesion molecule-1, VCAM-1, and peripheral lymph node addressin. Analysis of Tie2-green fluorescence protein transgenic mice demonstrated that CD56(bright) cells adhered primarily to vascular endothelium within the decidua basalis. Microdomain localization of adhesion involving large clusters of lymphocytes was induced on uteri from natural matings, but not pseudopregnancy. Steroid hormones also had independent effects on L-selectin function in splenic lymphocytes that mimicked physiological stimulation induced by pregnancy or fever-range temperatures. These results provide the first evidence for coordinated, organ-specific, steroid hormone-induced changes in lymphocyte homing mechanisms that could contribute to local and systemic immune responses during pregnancy.