Advances in infertility treatment had the most extraordinary breakthrough with the birth of the first in vitro fertilization baby in 1978. Fourteen years later, intracytoplasmic sperm injection has been introduced for the treatment of male factor infertility. Intra cytoplasmic sperm injection in combination with testicular sperm extraction has allowed men with azoospermia to father children. In fact, as long as a fully developed spermatozoon is identified, it can be utilized or can even be duplicated to inseminate several oocytes while providing information on its genomic content. There are, however, men who are suffering from spermatogenic arrest, where no post-meiotic germ cells are retrieved, and therefore, unable to generate their own offspring. More recently, the successful isolation and cultivation of spermatogonial stem cells has allowed the exploration of their biological characteristics and their application in therapeutic approaches following transplantation or in vitro maturation. Finally, men diagnosed with germ cell aplasia can only be treated by donor or de novo generated gametes. In the past several years, we have attempted to manufacture gametes by inducing haploidization of somatic cells and more recently, generating sperm-like cells through embryonic stem cell differentiation.

OBJECTIVE: To assess the role of environmental aggressors on imprinting marks of embryonic and extraembryonic tissues of conceptuses conceived by nuclear transplantation. DESIGN: Expression of imprinted genes in embryonic and extraembryonic tissues was measured and compared to those conceived in vivo or by somatic cell nuclear transfer. MATERIALS AND METHODS: Photoirradiation of mouse oocytes was used to induce cytoplasmic damage and NT as a model system for future correction. All fetuses were delivered by c/s on day 19 of gestation. In another experiment cloning was performed by injecting enucleated oocytes with somatic nuclei followed by chemical activation. Offspring derived from natural mating was used as control. qRT-PCR was performed with primers for the target sequence of Igf2, H19, and Igf2r genes. RESULTS: Induced maturation arrest in 445 GV oocytes was corrected in 51.2% by transplanting the nucleus into intact cytoplasts. Of these corrected oocytes, 59.9% were fertilized (139/232) and 132 developed as transferable embryos that yielded 17 fetuses. Of these, 15 became adults with a normal phenotype and able to reproduce. NT fetuses had an average weight of 1.7 gr and 1.5 for the control, while the mean placental weights were 0.35 and 0.13 gr, respectively. Of the 64 MII oocytes, 94% were successfully enucleated and 58 injected with a somatic nucleus. Of those, 38 displayed 2PN, with 11 developing as blastocysts. Cloned conceptuses did not develop past the blastocyst stage. A total of 8 cloned and equal number of control blastocysts were individually processed for epigenetic analysis. There was a concordant gene expression for all genes analyzed between embryonic (lung) and extraembryonic (placenta) tissues in the in vivo derived fetuses. In the NT specimen, the analysis of placental tissue evidenced a down regulation of H19 (P<0.001) and concurrent under expression of Igf2 (P=0.01), while the fetuses were unaffected. When cloned blastocysts were compared to unmanipulated counterpart, a 2.3 fold upregulation of H19 (P<0.001) and a 3.4 fold increase of Igf2r (P<0.001) were consistently measured fetuses. CONCLUSIONS: Parent-specific expression was maintained in embryonic and extraembyronic tissues of the in vivo conceived. The impact of embryo manipulation was identifiable in the placentas without affecting fetal imprinting marks. The unaltered gene expression of the manipulated fetuses translated in a normal pre- and post-implantation development that was instead compromised in the clones.

OBJECTIVE: To craft a database where a reproductive laboratory can store records concurrently and quickly provide precise information to each user. Here, we compared the efficiency and accuracy of such a customized database with that of a conventional hard copy and spreadsheet system. DESIGN: The Patient Demographics (PD) commences as the core and the main page of the database. From the PD a unique identifier branches out to 4 tables of andrological information as well as ICSI. From ICSI, another distinct identifier is used for 4 tables of IVF assessments. MATERIALS AND METHODS: A computer designated as the local area network acted as a server containing the database. All users participated in a 1 hr training class. Tests for both spreadsheet and study databases were run for all recurrent clinical and research scenarios. RESULTS: The user-friendly database included one main page PD and 5 tables: Semen Analysis (SA), IUI, Cryopreservation (Cryo), Cryostorage of Surgical Specimens (CSS), and ICSI. All tables can be accessed from PD in form of portals which allow unlimited storage of attempts per procedure, all viewable as a summary on one page. From Sept 2006 to May 2008, we entered 3470 PD, 2059 SA, 3509 IUI, 630 Cryo, 231 CSS, and 1878 ICSI cycles. The information for each table can be entered directly at the laboratory bench as soon as it becomes available. At the end of each procedure, the display print-out generated a hard copy for laboratory filing, and for patient charting. Regarding security, only specific personnel were granted privileges where a password had to be entered prior to gaining access or enter records into the database. The privileges for each password are tailored to the individual's needs. The likelihood of unintentionally altering results or leakage of patients' information is reduced. This database system drastically reduced the time required for technicians to transcribe, photocopy, classify, and enter a specific record. CONCLUSIONS: The conventional hard copy and spreadsheet is habitually vulnerable to inconsistencies. A customized database on the other hand, allows a single entry, provides data storage, and can be quickly modified according to the guidelines for standard operating procedures. The different levels of access grant a superior level of security. Lastly, because of the possibility to customize the screen similar to the original paper reports, it renders the use of a multi-relational database straightforward and pleasant for both medical and technical staff alike.

OBJECTIVE: To assess developmental potential of haploid mammalian embryos, we assess genome-wide DNA methylation of conceptuses generated by inseminating enucleated oocytes or by haploid parthenogenesis. DESIGN: The timing of development of mono-andric and mono-gynic conceptuses was analyzed. Epigenetic marks were assessed and compared with ICSI generated conceptuses. MATERIALS AND METHODS: To obtain mono-andric pseudo-embryos, enucleated mouse MII oocytes were injected with single sperm heads, while mono-gynic counterparts were generated by SrCl2 treatment. Cleavage of these constructs individually cultured was monitored at hourly interval up to 120. Some pseudo-embryos were processed for cytogenetic analysis at the second and third mitosis, while genome-wide DNA methylation patterns were detected by monoclonal antibodies raised against the methyl cytosine group. ICSI conceptuses served as control. RESULTS: Among the experimental groups, the proportion of constructs that reached the octet stage were comparable to the bi-parental counterparts. While the mono-gynic that reached the morula stage were comparable to the control, only 63.2% of the paternally derived constructs reach this stage. At the blastocyst stage, both mono-andric (15.8%) and mono-gynic (17.6%) development was remarkably compromised (P < 0.001), and required a longer time (P < 0.01). The delay in cleavage was prominent among the sperm-derived constructs. Karyotypic analysis revealed that pseudo-blastomeres of mono-andric at second (88.2%, 15/17) and third mitosis (91.3%, 21/23) confirmed their haploid status. The bi-parental embryos demonstrated active demethylation of the sperm nucleus earlier than the maternal genome, while both underwent progressive loss of methylation to regain it in synchrony at the blastocyst stage. Interestingly, the genome of mono-andric embryos failed to show any methylation changes until the octet stage, while mono-gynic maintained a methylation profile similar to the female genome of bi-parental embryos. CONCLUSIONS: We confirmed the compromised development of haploid embryos irrespective of gender. Differences in epigenetic marks as assessed by DNA methylation patterns are responsible for the restricted development of mono-andric embryos, possibly due to the absence or inactivation of the X chromosome.

OBJECTIVE: To investigate pregnancy outcome of cycles of IUI converted to IVF as well as IVF cycles with poor response converted to IUI taking into consideration couples characteristics and comparing them to ideal IUI cycles. DESIGN: Clinical pregnancy rates were compared with the 3 groups. MATERIALS AND METHODS: A fresh semen sample from their partners was analyzed for appropriate sperm characteristics. The sample processed by a single layer density gradient with an 80% recovery rate. Patients have documented patent tubes (one or both) by HSG study. Normal baseline u/s and serum FSH, LH and E2 levels were documented before treatment. COH started on day 2-3 of menses using 75-400 IU of recombinant or urinary FSH or hMG. Subsequent monitoring for serum E2, LH and transvaginal sonograms from day 7 thru 18 of cycles were performed and gonadotropins were titrated accordingly. When leading follicles measured 16-20mm in size, uhCG was administered intramuscularly to induce ovulation. The average maternal age for the general IUI population (n=674) was 35.3±5. Cancelled IVF (C-IVF; n=91) are patients that have low number of follicles with an average age of 39.6±3. Cancelled IUI (C-IUI; n=15) are patients that have at least 2 follicles with a maximum diameter of ≥20mm, whose average age was 34.9±5. RESULTS: The mean semen parameters were similar between IUI, C-IVF, and C-IUI with a concentration >50x106/ml, with ≥50% motility and 4% normal morphology. The C-IVF was older than IUI (P<0.001). The average number of attempts per patient was 1.9±1.3 for a standard IUI procedure and 1.1±0.3 for both C-IVF and C-IUI. A total of 132 IUI patients became pregnant, 8 after C-IVF, and 5 after C-IUI (P=0.01). When stratified according to maternal age <35, 35-39 and ≥40 years old, there was an inverse correlation between clinical pregnancy and maternal age in the standard IUI. On the other hand, C-IVF with patients less than 35 had 50% clinical pregnancy rate, 5.1% in 35-39, and 7.1% for patients ≥40, while the C-IUI follow the same trend as the standard IUI. CONCLUSIONS: In our center with standard IUI, we were still able to obtain a reasonable clinical outcome of 20%. However, some patients (C-IVF and C-IUI) still need to be "rescued" to avoid multiple gestations. Patients that are destined for IVF with good semen parameters and patent tubes can also be helped with an IUI cycle when there is a poor follicular development. Although requiring an adequate setting, IUIs can be successfully and safely converted to standard IVF.

OBJECTIVE: To assess the contribution of the male genome to the embryo's ability to develop and implant.DESIGN: Fertilization, embryo development, implantation and pregnancy outcomes were assessed in couples with a male partner suffering from spermatogenic failure and compared to normozoospermic couples.MATERIALS AND METHODS: ICSI cycles from Sept 1993 to Mar 2008 were reviewed and ejaculated and TESE cycles were identified. Only normal semen samples with a concentration of ≥20x106/ml, motility of ≥40%, and morphology ≥4% normal forms as well as NOA men undergoing TESE were included in the study. Female patients were then allocated according to the age groups as <35 or ≥35 yrs old. Fertilization, embryo cleavage and quality, and pregnancy outcome together with losses were compared.RESULTS: A total of 1652 ejaculated and 620 NOA patients were identified. When patients were categorized according to the maternal age and sperm source, women <35 yrs old inseminated with normozoospermic specimens (n=441) had a higher fertilization rate (79.1%) than the TESE (n=370) cycles (53.7%; P<0.001). The embryo quality and implantation rates were comparable between the two sperm sources (27.9 vs 30.6%). Furthermore, in spite of the similar number of embryos replaced, no differences were observed when clinical pregnancies (presence of a FHB) were compared (46.5 vs 44.6%). In addition, the incidence of pregnancy wastage for the ejaculate group was 4.9% (10/205) while for the NOA was 6.1% (10/165). When cycles with advanced maternal age (≥35 yrs old) were considered, the fertilization rate was similarly higher in the ejaculated cohort (76.7% vs 57.5; P<0.001) while, the embryo quality and the average number of conceptuses transferred were comparable between the two groups. Further, the clinical pregnancy rates were similar at 31.4% and 33.2% between the ejaculated and TESE sources. The proportion of embryos that implanted in the ejaculated group was 14.5% (536/3689) while in the TESE cohort, 17.2% (122/707). Interestingly, the rate of pregnancy losses was not higher in the TESE-treated patients (10.8%) than the normozoospermic group (15.5%).CONCLUSIONS: In contradiction to other studies implying that a suboptimal male gamete is responsible for poor embryo development and impaired implantation, we observed that men with compromised spermatogenesis reproduce similarly to their normozoospermic counterparts. From these findings, maternal age remains the sole culprit for the impaired clinical outcome. OBJECTIVE: To assess the contribution of the male genome to the embryo's ability to develop and implant. DESIGN: Fertilization, embryo development, implantation and pregnancy outcomes were assessed in couples with a male partner suffering from spermatogenic failure and compared to normozoospermic couples. MATERIALS AND METHODS: ICSI cycles from Sept 1993 to Mar 2008 were reviewed and ejaculated and TESE cycles were identified. Only normal semen samples with a concentration of ≥20x106/ml, motility of ≥40%, and morphology ≥4% normal forms as well as NOA men undergoing TESE were included in the study. Female patients were then allocated according to the age groups as <35 or ≥35 yrs old. Fertilization, embryo cleavage and quality, and pregnancy outcome together with losses were compared. RESULTS: A total of 1652 ejaculated and 620 NOA patients were identified. When patients were categorized according to the maternal age and sperm source, women <35 yrs old inseminated with normozoospermic specimens (n=441) had a higher fertilization rate (79.1%) than the TESE (n=370) cycles (53.7%; P<0.001). The embryo quality and implantation rates were comparable between the two sperm sources (27.9 vs 30.6%). Furthermore, in spite of the similar number of embryos replaced, no differences were observed when clinical pregnancies (presence of a FHB) were compared (46.5 vs 44.6%). In addition, the incidence of pregnancy wastage for the ejaculate group was 4.9% (10/205) while for the NOA was 6.1% (10/165). When cycles with advanced maternal age (≥35 yrs old) were considered, the fertilization rate was similarly higher in the ejaculated cohort (76.7% vs 57.5; P<0.001) while, the embryo quality and the average number of conceptuses transferred were comparable between the two groups. Further, the clinical pregnancy rates were similar at 31.4% and 33.2% between the ejaculated and TESE sources. The proportion of embryos that implanted in the ejaculated group was 14.5% (536/3689) while in the TESE cohort, 17.2% (122/707). Interestingly, the rate of pregnancy losses was not higher in the TESE-treated patients (10.8%) than the normozoospermic group (15.5%). CONCLUSIONS: In contradiction to other studies implying that a suboptimal male gamete is responsible for poor embryo development and impaired implantation, we observed that men with compromised spermatogenesis reproduce similarly to their normozoospermic counterparts. From these findings, maternal age remains the sole culprit for the impaired clinical outcome.

OBJECTIVE: To identify the predictability for a successful oocyte activation and consequent fertilization, spindle imaging was assessed on oocytes treated with different activators. DESIGN: In vitro matured oocytes were exposed to chemical or physical inducers. Mature oocytes were examined for meiotic spindle presence prior to oocyte treatment. MATERIALS AND METHODS: Spare germinal vesicle (GV) oocytes were obtained from consenting patients undergoing ICSI (IRB # 0198-082). GV oocytes were incubated in sequential medium for 24 hours to allow maturation to metaphase II (MII). Presence of meiotic spindles was assessed by a polarized microscope immediately prior to activation. MII oocytes with a first polar body (PB) were subjected to three different activators. The first group was exposed to 10 μM calcium ionophore (Ca-I) for 20 min. The second group (EP) was treated by electroporation (1.0 kV/cm, 130 μs) with custom microelectrodes (EggZyter®) in an electrolytic medium. Finally, the third group (Sr) was exposed to a Ca-free medium supplemented with 10 mM SrCl2 for 6 hours. Treated oocytes were subsequently cultured and assessed for PN formation and PB extrusion 16-20 hours later. Successful activation was confirmed once a clear PN and distinct PBs were visualized. RESULTS: A total of 55 GV oocytes were donated from 22 patients (33.6 ±5 years). The oocyte maturation rate was 65.4% (36/55). To establish the predictability, 11 MII oocytes were scanned for spindle visualization and 7 (63.6%) of those did actually display birefringent spindle structures. The activation rate following treatment of calcium ionophore of these oocytes with spindle present was 71.4% (5/7), while only 25.0% (1/4) in those without. In a subsequent group, only oocytes where spindle was seen (n=25) were allocated to different stimuli. While comparable activation was observed between Ca-I (53.8%, 7/13) and EP (66.7%, 4/6) groups, none were observed with SrCl2 (0/6, P < 0.05). CONCLUSIONS: To obtain successful oocyte activation for recurrent fertilization failure or somatic cell nuclear transfer procedures is preferable to confirm metaphase spindle presence. Calcium ionophore activation or electroporation prove comparable in achieving pronuclear formation and second polar body extrusion in more than half of oocytes.

OBJECTIVE: There is an optimal timing for fertilization and embryo cleavage to occur that often translates in a successful clinical outcome. However, it remains standard procedure in most laboratories to evaluate oocytes that fail to fertilize at the expected time. This study aims at determining whether it is still useful to evaluate these oocytes. DESIGN: A retrospective analysis of ICSI cycles where at least one oocyte displayed a delayed fertilization was carried out. Clinical outcome was compared with ICSI cycles displaying timely fertilization. MATERIALS AND METHODS: We analyzed 1,878 ICSI cycles between Jan 2007 to Mar 2008. For each cycle, we followed each oocyte from injection to transfer, identifying cases in which at least one oocyte showed delayed fertilization. Delayed fertilization is defined as the absence of pronuclei at 14-16 hrs post-injection, followed by the appearance of two pronuclei at 22-23 hrs. We compared fertilization and clinical pregnancy rates among cycles with delayed fertilization and those showing timely fertilization, controlling for differences in hCG administration, egg retrieval time and ICSI timing. Clinical pregnancy was defined as the presence of at least one FHB. RESULTS: The study group (n=1,878) was comprised of an average maternal age of 36.1 ± 6 years, an overall fertilization rate of 72.8% (10,913/14,990) and a clinical pregnancy rate of 30.9% (580/1,878). Out of 1,878 cycles, 107 (5.7%) had at least one oocyte showing delayed fertilization. Patients with mixed fertilization timing are listed in Table.Table 1Outcome of cycles with different fertilization timingFertilizationTimelyTimely & delayedCycles1,702101MII oocytes13,5701,037Fertilization rate (2PN)9,972 (73.5)745 (71.8)Late fertilization eggs0138Adjusted fertilization9,972 (73.5)a883 (85.1)aClinical pregnancy538 (31.6)b37 (36.6)baP< 0.0001, bP< 0.01. Open table in a new tab aP< 0.0001, bP< 0.01. The group including mixed fertilization displayed a significantly a higher fertilization rate and clinical pregnancy rate. In 30 of those patients, 37 late fertilized embryos were transferred. In order to prove that those zygotes can generate a pregnancy, we identified six patients that received exclusively those zygotes and observed one ongoing pregnancy. CONCLUSIONS: Although the occurrence of late fertilization is remarkably low, routine assessment for the delayed appearance of pronuclei proved to be a meaningful policy. The observed benefit on clinical outcome implies the need for sustaining the practice of identifying these zygotes.

OBJECTIVE: To assess whether the maternal DNA has any affect on the epigenetic marks of the sperm genome, we analyzed the methylation profiles of male nuclei in presence and absence of the female DNA. DESIGN: To assess genome-wide DNA methylation patterns of uni- and bi-parental conceptuses at fertilization and during early embryo development, haploid androgenones and reconstituted biparental embryos were generated by nuclear transfer. ICSI derived embryos served as control. MATERIALS AND METHODS: Following enucleation, mouse MII oocytes injected with a single sperm were cultured and allowed to cleave. To assess genome wide methylation, experimental constructs were exposed to monoclonal antibodies. Some karyoplasts isolated from the cleaving haploid androgenones were also transferred to parthenogenetically activated single pronuclear (PN) oocytes to prove their ability to function as male genome replicates. The constructs were electrofused to constitute a diploid zygote and cultured for up to 4 days. Post-implantation development and offspring wellbeing were also evaluated. RESULTS: Of 91 MII oocytes 95.6% survived enucleation and following single sperm injection displayed a single male PN. All of these 87 androgenones developed to pair, 75 (82.4%) to quartet, 59 (64.8%) to octet, 55 (60.4%) to morula, while only 3 (3.3%) cavitated. As expected, in ICSI zygotes the male PN lost methylation while the female genome remained methylated. Surprisingly haploid androgenones showed no active DNA demethylation at pseudo-PN stage and instead maintained the original methylation status up to the octet stage, and only from this stage onward passive demethylation occurred. A total of 87 karyoplasts were fused with activated oocytes to generate 85 (97.7%) zygotes that developed 68 (78.2%) blastocysts. Transfer of 30 blastocysts in pseudopregnant mice yielded 12 offspring (40.0%). Once the male karyoplasts were transplanted to the ooplasm in presence of the female genome they regained the ability to actively demethylate. CONCLUSIONS: Exposure of the male gamete to a mature ooplasm was not accompanied by loss of methylation prior to DNA replication. This methylated status allows the nucleus to replicate maintaining its developmental potential as a male gamete. However, replicated copies of the male genome promptly underwent demethylation once transplanted into female genome bearing ooplasms. This implies a role of female nucleus in modulating male genome epigenesis.

ESCs can differentiate into PGCs known to appear in the proximal epiblast of the egg cylinder by 6.5 dpc as soon as the BMP is expressed. Specific growth factors are essential in enhancing the appearance and survival of germ cells increasing their chances of steering them into meiosis. Here, by adding a cocktail of growth factors, we designed a favorable in vitro environment aimed at increasing PGC appearance among ESCs through embryoid body (EBs) development and attempted to correlate PGCs so derived with those observed in in vivo-derived mouse embryos. Mouse ESCs were maintained in a standard medium supplemented with LIF. EBs were generated in standard DMEM+serum, BMP4, or a combination of BMP4, 7, and 8b. At regular time intervals, cells were assessed for early germ cell specific markers, namely VASA, Fragilis, and c-Kit. Pre- (E6.5) and post- (E12.5) migratory PGCs were identified in embryos sectioned following vascular perfusion. Embryo slices were either 5 or 8 μm, depending on the age. Dissociation of EBs were sorted to enrich putative PGCs prior to culture into an in vitro maturation medium. The size of the embryos at E6.5 was about 15 × 22 μm. Approximately 3 sections (5 μm each) were obtained per embryo. A total of 14.6% (12/82) of the cells per embryo slice expressed fragilis – confirming premigratory PGCs. For E12.5 embryos, with a developed gonadal ridge estimated to be 25 × 87.5 μm, germ cell component of early post-migratory PGCs was depicted as 25.9% (45/174) by VASA expression. EB development was comparable in all media and after cell dissociation the cell number ranged from 550 to 3000. Spontaneous germ cell appearance on day 3 of culture was detected in standard medium at ∼1% (1/98). The presence of BMP4, increased PGC appearance to 36.2% (58/160) topping with the combination of the BMP family to 44.9% (92/205). Although there was an early and consistent appearance of PGC since day 3, they only self-renewed for up to 8 days and progressively declined thereafter. Allocation to the in vitro maturation medium failed to drive PGCs into meiosis. We confirm that germ cell differentiation from ESCs can be greatly enhanced by the action of specific growth factors. Morphology and marker presence mimicked their counterpart of epiblastic origin. Although we were able to enrich and stimulate self-renewal and proliferation of PGCs in early EBs, their spontaneous apoptosis impeded their further passage to later maturational stages.

Aiming to ease the ethical debates surrounding the ESC techniques, we have shown that microsurgical isolation of inner cell mass (ICM) cells is a feasible method to harvest embryonic stem cells (ESCs) maintaining embryo viability. Mouse embryonic fibroblasts (MEF) as a feeder cell layer have a paramount role for establishing ESCs, however, represent a source of xenogenic contamination, are labor intensive, and at times produce inconsistent results. Single cells were isolated from the ICM of mouse blastocysts. The efficiency of ESC derivation was compared between two different culture conditions according to presence or absence of adjuvant cell support. Pluripotency characteristics of the derived ESC lines were confirmed as well as pre- and post-implantation development of the biopsied embryos. Blastocysts were obtained by culturing zygotes, from mated mice after ovarian stimulation. Individual cells (3–5) were dislodged from the ICM and aspirated into a micropipette loaded with trypsin, and plated individually either on adjuvant cell system with conventional DMEM (MEF-D) or directly on a gelatin coated dish with ESGRO CompleteTM (ESGRO-C). Once ICM cells developed into epiblast-like aggregates, first dissociation was carried out by enzymatic digestion. Coincidentally, the biopsied embryos were transferred to pseudopregnant females. Intact blastocysts were used as controls. The ESC lines so derived were tested for pluripotency. Of a total of 119 blastocysts 46 were biopsied and 43 yielded isolated ICM cells that were allocated to either MEF-D (n = 30), or ESGRO-C (n = 16) system. The remaining 73 intact blastocysts were assigned to the same culture conditions serving as controls. Biopsied ICM cells formed 12 cell plaque aggregates and yielded 8 ESC colonies (23.5%) in MEF-D while no cell aggregates developed in ESGRO-C. ESC harvesting from intact control embryos in MEF-D was 17.6 and 9.1% in ESGRO-C, respectively. All the putative ESC lines showed pluripotent characteristics. Live birth rate of manipulated blastocysts was 43.3%, not different from the control (55.2%). The isolation of individual epiblast cells from blastocysts allows ESC harvesting of comparable quality to those derived from methodology sacrificing the entire embryo. Moreover, ICM biopsy did not impair embryo developmental competence. This study confirmed that individually isolated ICM cells necessitated adjuvant cells for stem cell generation.

ObjectiveThe scarcity of gametes often represents a main hindrance to overcoming spermatogenic failure through the use of ART. Thus, the possibility to propagate a male genome would provide an alternative means through which to consistently obtain conceptuses.DesignTo duplicate the sperm genome, haploid androgenotes were generated and allowed to cleave. Haploid parthenotes were obtained by activating eggs. To assess the ability of the male clones to support normal embryo development, diploid constructs were derived by transferring them into parthenotes.Materials and methodsFollowing enucleation, mouse MII oocytes injected with a single sperm and defined as androgenic replicates, were cultured for ∼20 h. To generate gynogenic counterparts, another cohort of eggs was exposed to SrCl2. Karyoplasts isolated from the cleaving haploid androgenotes were transferred to haploid parthenotes. The constructs were electrofused to constitute a diploid zygote and cultured for 96 h. Some of the haploid androgenotes were processed for cytogenetics. ICSI conceptuses served as control. To investigate post-implantation development and offspring wellbeing, blastocysts were transferred to pseudo-pregnant mice.ResultsOf 192 MII oocytes initially manipulated 168 survived, and all came to display a single male PN. A total of 155 cleaved and maintained their haploid status in 88% analyzed. Oocyte activation was successful in 95% and once constructs were electrofused, 98% generated biparental zygotes. During the 4 day culture, 77% of the study constructs developed into blastocysts at a rate comparable to ICSI embryos (81%). However, the transfer of 64 blastocysts yielded only 11 offspring (17%), fewer than the 43% achieved in the control group (P<0.05). Moreover, of those only 4 grew normally whereas all the ICSI offspring became adults. Although 3 were cannibalized and 4 died soon after birth, no genotypic or phenotypic anomalies were identified in the study group.ConclusionsIt is possible to replicate the male genome through its injection into ooplasts. Such androgenotes maintain their genotype, ploidy, and the ability to achieve syngamy. Moreover, their propagation as cleaving pseudo-blastomeres did not alter epigenetic imprinting of the male gamete. In comparison to whole genome cloning, the relatively higher reproducibility of the technique indicates that it is possible to create multiple copies of the male genome through which to gain genetic information on a particular gamete or to propagate it when scarce. ObjectiveThe scarcity of gametes often represents a main hindrance to overcoming spermatogenic failure through the use of ART. Thus, the possibility to propagate a male genome would provide an alternative means through which to consistently obtain conceptuses. The scarcity of gametes often represents a main hindrance to overcoming spermatogenic failure through the use of ART. Thus, the possibility to propagate a male genome would provide an alternative means through which to consistently obtain conceptuses. DesignTo duplicate the sperm genome, haploid androgenotes were generated and allowed to cleave. Haploid parthenotes were obtained by activating eggs. To assess the ability of the male clones to support normal embryo development, diploid constructs were derived by transferring them into parthenotes. To duplicate the sperm genome, haploid androgenotes were generated and allowed to cleave. Haploid parthenotes were obtained by activating eggs. To assess the ability of the male clones to support normal embryo development, diploid constructs were derived by transferring them into parthenotes. Materials and methodsFollowing enucleation, mouse MII oocytes injected with a single sperm and defined as androgenic replicates, were cultured for ∼20 h. To generate gynogenic counterparts, another cohort of eggs was exposed to SrCl2. Karyoplasts isolated from the cleaving haploid androgenotes were transferred to haploid parthenotes. The constructs were electrofused to constitute a diploid zygote and cultured for 96 h. Some of the haploid androgenotes were processed for cytogenetics. ICSI conceptuses served as control. To investigate post-implantation development and offspring wellbeing, blastocysts were transferred to pseudo-pregnant mice. Following enucleation, mouse MII oocytes injected with a single sperm and defined as androgenic replicates, were cultured for ∼20 h. To generate gynogenic counterparts, another cohort of eggs was exposed to SrCl2. Karyoplasts isolated from the cleaving haploid androgenotes were transferred to haploid parthenotes. The constructs were electrofused to constitute a diploid zygote and cultured for 96 h. Some of the haploid androgenotes were processed for cytogenetics. ICSI conceptuses served as control. To investigate post-implantation development and offspring wellbeing, blastocysts were transferred to pseudo-pregnant mice. ResultsOf 192 MII oocytes initially manipulated 168 survived, and all came to display a single male PN. A total of 155 cleaved and maintained their haploid status in 88% analyzed. Oocyte activation was successful in 95% and once constructs were electrofused, 98% generated biparental zygotes. During the 4 day culture, 77% of the study constructs developed into blastocysts at a rate comparable to ICSI embryos (81%). However, the transfer of 64 blastocysts yielded only 11 offspring (17%), fewer than the 43% achieved in the control group (P<0.05). Moreover, of those only 4 grew normally whereas all the ICSI offspring became adults. Although 3 were cannibalized and 4 died soon after birth, no genotypic or phenotypic anomalies were identified in the study group. Of 192 MII oocytes initially manipulated 168 survived, and all came to display a single male PN. A total of 155 cleaved and maintained their haploid status in 88% analyzed. Oocyte activation was successful in 95% and once constructs were electrofused, 98% generated biparental zygotes. During the 4 day culture, 77% of the study constructs developed into blastocysts at a rate comparable to ICSI embryos (81%). However, the transfer of 64 blastocysts yielded only 11 offspring (17%), fewer than the 43% achieved in the control group (P<0.05). Moreover, of those only 4 grew normally whereas all the ICSI offspring became adults. Although 3 were cannibalized and 4 died soon after birth, no genotypic or phenotypic anomalies were identified in the study group. ConclusionsIt is possible to replicate the male genome through its injection into ooplasts. Such androgenotes maintain their genotype, ploidy, and the ability to achieve syngamy. Moreover, their propagation as cleaving pseudo-blastomeres did not alter epigenetic imprinting of the male gamete. In comparison to whole genome cloning, the relatively higher reproducibility of the technique indicates that it is possible to create multiple copies of the male genome through which to gain genetic information on a particular gamete or to propagate it when scarce. It is possible to replicate the male genome through its injection into ooplasts. Such androgenotes maintain their genotype, ploidy, and the ability to achieve syngamy. Moreover, their propagation as cleaving pseudo-blastomeres did not alter epigenetic imprinting of the male gamete. In comparison to whole genome cloning, the relatively higher reproducibility of the technique indicates that it is possible to create multiple copies of the male genome through which to gain genetic information on a particular gamete or to propagate it when scarce.

Sperm immobilization prior to injection represents the only pre-treatment for a successful ICSI procedure. However, mechanical immobilization is a rather crude and often inconsistent way of disrupting the sperm membrane. To identify a method that disrupts and destabilizes the sperm membrane more consistently we exposed human spermatozoa to a synthetic lysolipid compound, lysolecichin (LLT), before injecting them into mouse and human oocytes and compared them to the standard method. Ejaculated spermatozoa were obtained from consenting patients undergoing ART treatment and processed in the standard manner. The effect of LLT was titrated at 0.01, 0.02, 0.05, and 0.1% concentrations to identify the minimal amount required to exert a satisfactory stripping as monitored by the loss of motility and confirmed by transmission electron microscopy. MII oocytes obtained from superovulated mice, were each piezo-injected with a single spermatozoon which had either been mechanically immobilized, or treated with LLT. Oocytes were observed for extrusion of the second polar body and for pronuclei (PNs). Human oocytes donated by consenting ICSI patients that had matured in vitro were injected with LLT- treated or mechanically-immobilized spermatozoa. The average time required for sperm immobilization by LLT was 4.7 sec with 0.1%, 8.9 sec with 0.05%, and 28.9 sec with 0.02%. No loss of motility was observed below 0.02%. Thus there was an inverse correlation between the LLT concentration and time required for the loss of sperm motility (R −0.63; P<0.01). The mouse oocyte 2PN rate was 90% following the injection of chemically treated spermatozoa, not different from mechanically immobilized spermatozoa (81.3%). However, mouse oocytes injected with intact motile human spermatozoa displayed 2PNs only in 6.7% (P<0.001). The injection of 14 human oocytes with spermatozoa exposed solely to 0.05% LLT resulted in fertilization only in 14.3% (2/14), clearly lower than with standard immobilization (48.6%, 18/37; P<0.05). This study confirmed the important role of sperm membrane treatment prior to ICSI for consistent PN formation. Although LLT provided a reliable, fast, and comparable membrane damage, human chemical immobilization of spermatozoa did not appear to offer any advantage over the more conventional mechanical immobilization method.

ObjectiveTo overcome the scarcity of spermatozoa in some infertile men, we have successfully cloned the male genome in the mouse. However, offspring survival has been inconsistent for reasons that are still unclear.DesignWe attempted to determine the ideal cell cycle by selecting different times at which to transplant the cloned male genomes into parthenotes in order to achieve syngamy and obtain pre- and post-implantation embryo development.Materials and methodsMale genome clones were generated by injecting spermatozoa into MII ooplasts retrieved from 7–11 week superovulated mice. Androgenetic pronuclei appearing consistently at 6 hrs after injection were presumed to be at the beginning of the S phase. This was considered the starting time at which to induce the generation of parthenotes that were scheduled at II, IV, and VI hr time intervals. The recipient parthenotes displayed a single pronucleus 6 hrs after exposure to SrCl2. Androgenetic pseudo-blastomeres cleaved about 12 hrs after the appearance of the first single pronucleus. This was considered the starting time for the time table of transplantation performed also at II, IV, and VI hr intervals. Fully-developed blastocysts were transferred to pseudo-pregnant mice with later monitoring of post-implantation development and of the wellbeing of any offspring.ResultsThe course of enucleation, activation, and electrofusion did not differ among the three experimental time plans. However, the IV hr approach generated 93.5% of blastocysts, followed by 88.5% in the II hr, and 75.9% in the VI hr plans. The actual live birth rate was similar in the three groups, being 12.5% (2/16), 26.3% (5/19), and 13.8% (4/29), respectively. However, a majority of the offspring (7 out of 11) died immediately after the birth. Nonetheless, in the IV hr experimental plan 4 out of 5 offspring are still healthy now at 6 weeks of age. The deceased offspring not cannibalized by the foster mothers, did not reveal any cytogenetic or histopathologic abnormalities.ConclusionsThese findings indicate that a specific pronuclear phase window needs to be selected in order to maintain parental pronuclear synchrony. This window, presumed to fall at the beginning of the S phase, allows syngamy and later normal post-implantation development of embryos fathered by male clones. ObjectiveTo overcome the scarcity of spermatozoa in some infertile men, we have successfully cloned the male genome in the mouse. However, offspring survival has been inconsistent for reasons that are still unclear. To overcome the scarcity of spermatozoa in some infertile men, we have successfully cloned the male genome in the mouse. However, offspring survival has been inconsistent for reasons that are still unclear. DesignWe attempted to determine the ideal cell cycle by selecting different times at which to transplant the cloned male genomes into parthenotes in order to achieve syngamy and obtain pre- and post-implantation embryo development. We attempted to determine the ideal cell cycle by selecting different times at which to transplant the cloned male genomes into parthenotes in order to achieve syngamy and obtain pre- and post-implantation embryo development. Materials and methodsMale genome clones were generated by injecting spermatozoa into MII ooplasts retrieved from 7–11 week superovulated mice. Androgenetic pronuclei appearing consistently at 6 hrs after injection were presumed to be at the beginning of the S phase. This was considered the starting time at which to induce the generation of parthenotes that were scheduled at II, IV, and VI hr time intervals. The recipient parthenotes displayed a single pronucleus 6 hrs after exposure to SrCl2. Androgenetic pseudo-blastomeres cleaved about 12 hrs after the appearance of the first single pronucleus. This was considered the starting time for the time table of transplantation performed also at II, IV, and VI hr intervals. Fully-developed blastocysts were transferred to pseudo-pregnant mice with later monitoring of post-implantation development and of the wellbeing of any offspring. Male genome clones were generated by injecting spermatozoa into MII ooplasts retrieved from 7–11 week superovulated mice. Androgenetic pronuclei appearing consistently at 6 hrs after injection were presumed to be at the beginning of the S phase. This was considered the starting time at which to induce the generation of parthenotes that were scheduled at II, IV, and VI hr time intervals. The recipient parthenotes displayed a single pronucleus 6 hrs after exposure to SrCl2. Androgenetic pseudo-blastomeres cleaved about 12 hrs after the appearance of the first single pronucleus. This was considered the starting time for the time table of transplantation performed also at II, IV, and VI hr intervals. Fully-developed blastocysts were transferred to pseudo-pregnant mice with later monitoring of post-implantation development and of the wellbeing of any offspring. ResultsThe course of enucleation, activation, and electrofusion did not differ among the three experimental time plans. However, the IV hr approach generated 93.5% of blastocysts, followed by 88.5% in the II hr, and 75.9% in the VI hr plans. The actual live birth rate was similar in the three groups, being 12.5% (2/16), 26.3% (5/19), and 13.8% (4/29), respectively. However, a majority of the offspring (7 out of 11) died immediately after the birth. Nonetheless, in the IV hr experimental plan 4 out of 5 offspring are still healthy now at 6 weeks of age. The deceased offspring not cannibalized by the foster mothers, did not reveal any cytogenetic or histopathologic abnormalities. The course of enucleation, activation, and electrofusion did not differ among the three experimental time plans. However, the IV hr approach generated 93.5% of blastocysts, followed by 88.5% in the II hr, and 75.9% in the VI hr plans. The actual live birth rate was similar in the three groups, being 12.5% (2/16), 26.3% (5/19), and 13.8% (4/29), respectively. However, a majority of the offspring (7 out of 11) died immediately after the birth. Nonetheless, in the IV hr experimental plan 4 out of 5 offspring are still healthy now at 6 weeks of age. The deceased offspring not cannibalized by the foster mothers, did not reveal any cytogenetic or histopathologic abnormalities. ConclusionsThese findings indicate that a specific pronuclear phase window needs to be selected in order to maintain parental pronuclear synchrony. This window, presumed to fall at the beginning of the S phase, allows syngamy and later normal post-implantation development of embryos fathered by male clones. These findings indicate that a specific pronuclear phase window needs to be selected in order to maintain parental pronuclear synchrony. This window, presumed to fall at the beginning of the S phase, allows syngamy and later normal post-implantation development of embryos fathered by male clones.

Polyvinylpyrrolidone (PVP) is often used for selection, immobilization and handling of spermatozoa during ICSI. PVP is a synthetic copolymer of roughly 360 kD, often used as a 10% concentration. It produces a deceleration of sperm motility, and thereby allows the selection of viable spermatozoa displaying a good morphology, facilitating their immobilization as well as providing good control of the sperm aspirated into the ICSI needle. PVP preparation involves mixing of the powder, filtration of the resulting viscous solution and finally adjusting its osmolarity. Also, it is suggested that the compound be purified by a dialysis that ensures its RNAse/DNAse-free condition. ICSI outcomes, such as fertilization characteristics, embryo cleavage, and pregnancy rates were compared in cases where a commercially available PVP at different concentrations vs. these outcomes using an in-house PVP preparation. The evaluations involved ICSI cycles with ejaculated spermatozoa. Cycles were assigned according to PVP preparations used, i.e. a home-made 10% (C) vs. commercially available 7% (S-7) and 10% (S-10) concentrations. Ovarian stimulation and the ICSI procedure were performed in a standard fashion. Fertilization was assessed 16–18 hrs later, with two consecutive elevated serum βhCG levels were observed 14 days following embryo transfer, and clinical pregnancy was defined by at least one fetal heartbeat. Of a total of 399 ICSI cycles, home-made PVP (C) was used for 297 cycles, 7% PVP (S-7) for 64, and 10% (S-10) for 38 cycles. The mean maternal age, semen parameters, mean number of oocytes injected, oocyte survival, and abnormal fertilization patterns were comparable among the three groups. S-7 had a fertilization rate of 69.2% - lower than groups C of 76.0% and S-10 of 77.8% (P<0.01). The embryonic cleavage rates were 79.9 (S-7), 82.6 (C), and 87.6% (S-10) (P<0.01). The mean number of embryos transferred on day 3 and day 5 was similar in all groups, as was the pregnancy rate. A 10% commercially available PVP seems as effective as 10% home-made PVP, and marginally superior to the commercial 7% PVP, in terms of fertilization and the pregnancies achieved. Thus it seems both useful and justified to use the ready-made PVP for sperm suspension in the ICSI protocol.

Approximately 10% of infertile men are azoospermic and the large majority of this group exhibit associated genetic disorders that include aneuploidy or chromosomal structural rearrangements such as micro-satellite deletions. Among the abnormalities, gonosomal problems are the most recurrent, with Klinefelter’s syndrome, in its complete or mosaic form responsible for severe spermatogenic failure. Here we analyze the clinical outcome of ART cases involving gonosomal trisomy. Fertilization, pregnancy characteristics, and perinatal outcome were evaluated in comparison to those of other NOA patients. To assess the effect on ICSI cycles of spermatogenic failure in the case of Klinefelter patients, we analyzed the patterns of fertilization, pregnancy as well as neonatal characteristics, as compared to these parameters in TESE cycles (control). Testicular tissue was collected by micro-TESE and processed for ICSI. Once spermatozoa were found, MII stage oocytes were fertilized by ICSI. Motility enhancers were employed where the spermatozoa so recovered were immotile. The fertilization pattern was recorded 12-17 hrs after ICSI and ultimately, pregnancy was confirmed by the presence of at least one fetal heartbeat. Klinefelter patients were offered sperm aneuploidy screening, preimplantation genetic diagnosis, and advice in regard to undergoing prenatal diagnosis. Among a total of 52 testicular biopsies obtained from patients (33.5 ± 7 yrs) with Klinefelter syndrome in 56 ICSI cycles from December 1995 to March 2006, 36 (69.2%) were revealed to have spermatozoa. A total of 56 TESE that included 8 frozen samples were utilized for ICSI. These samples had a mean concentration of 400,000/ml, motility of 1.4% ± 5, and no normal forms. The average number of oocytes retrieved was 12.5 (n = 781). The fertilization rate was 54.1% (322/595) with an abnormal fertilization (1 and 3PN) ranging between 2.6 and 5.7%. In a total of 48 cycles at least one conceptus was replaced resulting in a clinical pregnancy rate (FHB) of 30.3% (17/56) and a delivery of 26.8% (15/56). These included 8 singletons, 4 twins, and 3 triplets, totaling 25 children (12 boys and 13 girls), in which no malformations were observed. . In NOA patients with normal karyotypes, serving as controls, the fertilization was 56.9% and the clinical pregnancy rate was 31.6%. In this group, the deliveries and ongoing pregnancies together equaled 35.8% and resulted in 185 children with only two malformations (one major and one minor). A selective reduction because of a 47,XY+18 was performed in one set of twins. Spermatozoa obtained from testicular biopsies of Klinefelter patients performed in a similar manner to those of NOA patients. Even so, because of concerns related to sperm aneuploidy or meiotic defects, these cases should be offered sperm aneuploidy screening, PGD, and prenatal diagnosis.

The development of cloned embryos is variable and often limited. The failure to develop to term is most likely a function of incomplete or improper nuclear reprogramming that is reflected in an aberrant pattern of gene expression. Nonetheless, in spite of their impaired developmental ability it is still possible to derive ESCs from these embryos. Chromatin remodeling in somatic cells partly modulates their gene expression and is one of the prerequisites for nuclear reprogramming required to support early embryo development. The effect of chromatin treatment on the ability to generate cloned embryos and support their complete preimplantation development and ability to produce ESCs was assessed. Gene expression patterns of cloned blastocyst and the effect of these patterns on the ESC harvest was also evaluated. Fibroblasts were prepared by culturing minced skin tissue obtained from a male mouse. Mitotic extract was prepared by synchronizing cultured kidney cells at mitosis followed by their lysis. After permeabilization, the fibroblasts were exposed to mitotic extract with an ATP system, and their membrane resealed. MII oocytes were enucleated, then injected with either pretreated or intact single fibroblasts. Activated oocytes displaying 2PN were further cultured, while blastocysts generated from in vivo zygotes, and cultured similarly, served as controls. Total RNA was extracted and subjected to qRT-PCR to assess expression of Igf2, H19, and Igf2r, or for a whole genome chip analysis. Other blastocysts were plated for ESC harvesting and their characteristics were tested by pluripotency marker expression and by their differentiation capability in vivo and in vitro and epigenetic status of ESCs was evaluated as well. Among the MII cytoplasts injected conventionally with fibroblast nuclei, 28 of 30 survived, 21 displayed 2PN, and 3 (16.7%) reached the blastocyst stage. Among 36 ooplasts, 32 were successfully reconstituted by chromatin treatment and became 2PN zygotes, and 18 (50%) formed blastocysts. The latter showed a higher blastocyst formation rate (P < 0.05). Chip analysis revealed some aberrant gene expression patterns in the cloned blastocysts both with conventional NT and after chromatin treatment in comparison to their in vivo-fertilized counterparts. However, when the imprinted genes were assessed the cloned blastocysts developing after chromatin transfer had a pattern closer to that in those fertilized in vivo. When blastocysts generated from conventional NT were plated onto a feeder layer, 2 of 5 (40.0%) produced ESC lines. Two ESC lines were obtained after plating 4 blastocysts derived following chromatin treatment. By comparison, plating of 35 in vivo derived blastocysts generated 7 (20.0%) ESC lines. The ESC harvest rate was comparable among all the three groups, their pluripotency being confirmed by marker activity for over 10 passages. All ESC lines were able to differentiate in vitro and in vivo. Prior treatment of the transferred nuclei enhanced the rate of cloned blastocyst formation. Although overall gene expression patterns following somatic nuclear treatment were substantially unchanged, the expression of some key embryo developmental genes was similar to that of in vivo derived blastocysts. The ability to efficiently harvest ESCs with typical characteristics from those suboptimal embryos was not reduced by treatment of somatic nucleus.