Fertilization and Cleavage Axes Differ In Primates Conceived By Conventional (IVF) Versus Intracytoplasmic Sperm Injection (ICSI).

With nearly ten million babies conceived globally, using assisted reproductive technologies, fundamental questions remain; e.g., How do the sperm and egg DNA unite? Does ICSI have consequences that IVF does not? Here, pronuclear and mitotic events in nonhuman primate zygotes leading to the establishment of polarity are investigated by multidimensional time-lapse video microscopy and immunocytochemistry. Multiplane videos after ICSI show atypical sperm head displacement beneath the oocyte cortex and eccentric para-tangential pronuclear alignment compared to IVF zygotes. Neither fertilization procedure generates incorporation cones. At first interphase, apposed pronuclei align obliquely to the animal-vegetal axis after ICSI, with asymmetric furrows assembling from the male pronucleus. Furrows form within 30° of the animal pole, but typically, not through the ICSI injection site. Membrane flow drives polar bodies and the ICSI site into the furrow. Mitotic spindle imaging suggests para-tangential pronuclear orientation, which initiates random spindle axes and minimal spindle:cortex interactions. Parthenogenetic pronuclei drift centripetally and assemble astral spindles lacking cortical interactions, leading to random furrows through the animal pole. Conversely, androgenotes display cortex-only pronuclear interactions mimicking ICSI. First cleavage axis determination in primates involves dynamic cortex-microtubule interactions among male pronuclei, centrosomal microtubules, and the animal pole, but not the ICSI site.

Sperm deoxyribonucleic acid fragmentation assessment in normozoospermic male partners of couples with unexplained recurrent pregnancy loss: a prospective study.

OBJECTIVE: To determine whether sperm DNA integrity in normozoospermic male partners plays a role in idiopathic recurrent pregnancy loss (RPL). DESIGN: Prospective, cohort study. SETTING: Academic tertiary care center. PATIENT(S): Group I: 26 male partners of women with unexplained RPL. Group II: 31 normozoospermic males with proven fertility. INTERVENTION(S): Semen samples were collected by masturbation after 48-72 hours of abstinence. After liquefaction at room temperature, semen analysis was performed according to World Health Organization standards. Only samples with >20 × 10(6) spermatozoa/mL with at least 50% progressive sperm motility and 30 % normal morphology were selected for the study. DNA fragmentation of the sperm was assessed with TUNEL assay followed by flow cytometric analysis. MAIN OUTCOME MEASURE(S): Sperm DNA fragmentation in both groups. RESULT(S): Mean DNA fragmentation (mean ± SD) was significantly more in men with RPL (36.8 ± 5) compared with controls (9.4 ± 2.7). CONCLUSION(S): Sperm DNA fragmentation may play a role in unexplained RPL despite normal semen analysis parameters.

Interspecies chimera between primate embryonic stem cells and mouse embryos: monkey ESCs engraft into mouse embryos, but not post-implantation fetuses.

Unequivocal evidence for pluripotency in which embryonic stem cells contribute to chimeric offspring has yet to be demonstrated in human or nonhuman primates (NHPs). Here, rhesus and baboons ESCs were investigated in interspecific mouse chimera generated by aggregation or blastocyst injection. Aggregation chimera produced mouse blastocysts with GFP-nhpESCs at the inner cell mass (ICM), and embryo transfers (ETs) generated dimly-fluorescencing abnormal fetuses. Direct injection of GFP-nhpESCs into blastocysts produced normal non-GFP-fluorescencing fetuses. Injected chimera showed >70% loss of GFP-nhpESCs after 21 h culture. Outgrowths of all chimeric blastocysts established distinct but separate mouse- and NHP-ESC colonies. Extensive endogenous autofluorescence compromised anti-GFP detection and PCR analysis did not detect nhpESCs in fetuses. NhpESCs localize to the ICM in chimera and generate pregnancies. Because primate ESCs do not engraft post-implantation, and also because endogenous autofluorescence results in misleading positive signals, interspecific chimera assays for pluripotency with primate stem cells is unreliable with the currently available ESCs. Testing primate ESCs reprogrammed into even more naïve states in these inter-specific chimera assays will be an important future endeavor.

Derivation and characterization of novel nonhuman primate embryonic stem cell lines from in vitro-fertilized baboon preimplantation embryos.

The development of nonhuman primate (NHP) embryonic stem cell (ESC) models holds great promise for cell-mediated treatment of debilitating diseases and to address numerous unanswered questions regarding the therapeutic efficacy of ESCs while supplanting ethical considerations involved with human studies. Here we report successful establishment and characterization of 3 novel baboon (Papio cynocephalus) ESC lines from the inner cell mass of intracytoplasmic sperm injection-derived blastocysts. Embryos were cultured in an improved baboon embryo in vitro culture protocol. The inner cell mass of blastocyst was laser-dissected and plated on mouse embryonic fibroblast feeder cell monolayer in the NHP ESC culture medium. Three cell lines with characteristic ESC morphology have been cultured through an extended period (>14 months), with 2 male cell lines (UT-1 and -2) and 1 female cell line (UT-3) displaying normal baboon karyotypes. Reverse transcription-polymerase chain reaction analysis confirmed that all 3 lines express primate ESC pluripotency markers, including OCT-4, NANOG, SOX-2, TERT, TDGF, LEFTYA, and REX-1. All 3 lines demonstrated positive immunocytochemical staining for OCT-4, stage-specific embryonic antigen-3, stage-specific embryonic antigen-4, TRA-1-60, and TRA-1-81. Baboon ESCs injected into NOD/SCID mice formed teratomas with all 3 germ layers. In addition, embryoid body-like spherical structures were derived and initial outgrowth was observed when embedded into extracellular matrix Matrigel. The ESC lines established in this NHP model have the potential to extend our knowledge in the fields of developmental biology, regenerative medicine, and future applications, including preclinical safety assessment of in vivo stem cell therapy.

Assisted Reproductive Technologies (ART) with baboons generate live offspring: a nonhuman primate model for ART and reproductive sciences.

Human reproduction has benefited significantly by investigating nonhuman primate (NHP) models, especially rhesus macaques. To expand the Old World monkey species available for human reproductive studies, we present protocols in baboons, our closest Old World primate relatives, for assisted reproductive technologies (ART) leading to live born offspring. Baboons complement rhesus by confirming or modifying observations generated in humans often obtained by the study of clinically discarded specimens donated by anonymous infertility patient couples. Here, baboon ART protocols, including oocyte collection, in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), preimplantation development to blastocyst stage, and embryo transfer techniques are described. With baboon ART methodologies in place, motility during baboon fertilization was investigated by time-lapse video microscopy (TLVM). The first ART baboons produced by ICSI, a pair of male twins, were delivered naturally at 165 days postgestation. Genetic testing of these twins confirmed their ART parental origins and demonstrated that they are unrelated fraternal twins not identicals. These results have implications for ART outcomes, embryonic stem cell (ESC) derivation, and reproductive sciences.

Establishment and characterization of baboon embryonic stem cell lines: an Old World Primate model for regeneration and transplantation research.

Here we have developed protocols using the baboon as a complementary alternative Old World Primate to rhesus and other macaques which have severe limitations in their availability. Baboons are not limited as research resources, they are evolutionarily closer to humans, and the multiple generations of pedigreed colonies which display complex human disease phenotypes all support their further optimization as an invaluable primate model. Since neither baboon-assisted reproductive technologies nor baboon embryonic stem cells (ESCs) have been reported, here we describe the first derivations and characterization of baboon ESC lines from IVF-generated blastocysts. Two ESCs lines (BabESC-4 and BabESC-15) display ESC morphology, express pluripotency markers (Oct-4, hTert, Nanog, Sox-2, Rex-1, TRA1-60, TRA1-81), and maintain stable euploid female karyotypes with parentage confirmed independently. They have been grown continuously for >430 and 290 days, respectively. Teratomas from both lines have all three germ layers. Availabilities of these BabESCs represent another important resource for stem cell biologists.

Pedigreed primate embryonic stem cells express homogeneous familial gene profiles.

Human embryonic stem cells (hESCs) hold great biomedical promise, but experiments comparing them produce heterogeneous results, raising concerns regarding their reliability and utility, although these variations may result from their disparate and anonymous origins. To determine whether primate ESCs have intrinsic biological limitations compared with mouse ESCs, we examined expression profiles and pluripotency of newly established nonhuman primate ESC (nhpESCs). Ten pedigreed nhpESC lines, seven full siblings (fraternal quadruplets and fraternal triplets), and nine half siblings were derived from 41 rhesus embryos; derivation success correlated with embryo quality. Each line has been growing continuously for approximately 1 year with stable diploid karyotype (except for one stable trisomy) and expresses in vitro pluripotency markers, and eight have already formed teratomas. Unlike the heterogeneous gene expression profiles found among hESCs, these nhpESCs display remarkably homogeneous profiles (>97%), with full-sibling lines nearly identical (>98.2%). Female nhpESCs express genes distinct from their brother lines; these sensitive analyses are enabled because of the very low background differences. Experimental comparisons among these primate ESCs may prove more reliable than currently available hESCs, since they are akin to inbred mouse strains in which genetic variables are also nearly eliminated. Finally, contrasting the biological similarities among these lines with the heterogeneous hESCs might suggest that additional, more uniform hESC lines are justified. Taken together, pedigreed primate ESCs display homogeneous and reliable expression profiles. These similarities to mouse ESCs suggest that heterogeneities found among hESCs likely result from their disparate origins rather than intrinsic biological limitations with primate embryonic stem cells.