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Aneuploidy free oocytes may be pre-selected by testing the first and second polar bodies removed from oocytes following their maturation and fertilization. We present here our experience on the application of the method in IVF cycles from patients of advanced maternal age. Overall, 5590 oocytes were obtained from 917 cycles and tested by polar body sampling and fluorescent in situ hybridization (FISH) analysis using specific probes for chromosomes 13,16,18,21 and 22. FISH results were available in 4599 (82.2%) of 5590 oocytes studied, from which 2077(45.2%) were with aneuploidies. Thirty six point one percent of aneuploidies were of the first meiotic origin, and 29.3% of the second meiotic origin. Most errors in the first meiotic division were represented by chromatid errors. The transfer of embryos deriving from 2014 of 2520 aneuploidy free oocytes in 821 treatment cycles resulted in 182 (22.2%) clinical pregnancies and 140 healthy children born after confirmation of the polar body diagnosis. Polar body testing of oocytes provides an approach for pre-selection of aneuploidy free embryos, improving pregnancy rate in IVF patents of advanced maternal age.
Mol Cell Endocrinol 2001 Oct 22
PMID:Chromosomal abnormalities in the first and second polar body. 1157 32

Preimplantation genetic diagnosis (PGD) of translocations can be achieved through a variety of methods. For female carriers, a possibility is by polar body biopsy and analysis of its metaphase chromosomes using painting probes. For male carriers or female carriers with terminal breakpoints, metaphase chromosomes can also be studied by fusing blastomeres to enucleated oocytes. Otherwise, interphase analysis of the translocation can be performed using distal, subtelomeric or breakpoint spanning probes. The results obtained after PGD of translocations indicate a significant decrease in spontaneous abortions after the procedure, a good selection against unbalanced oocytes and embryos, and pregnancy rates that depend on the type of translocation involved. Balanced translocations occur in 0.2% of the neonatal population, but at a higher rate among infertile couples and patients with recurrent abortions. In a recent report, balanced translocations were found in 0.6% of infertile couples, 3.2% of couples that failed over ten IVF cycles, and 9.2% among fertile couples experiencing three or more consecutive first-trimester abortions (Hum. Reprod. 14 (1999) 2097). They were also found in 2-3.2% of males requiring ICSI (Hum. Reprod. 11 (1996) 2609; Hum. Reprod. 13 (1998) 576). PGD can be offered to carriers of balanced translocations as an alternative to prenatal diagnosis and pregnancy termination of unbalanced fetuses. In recent years, PGD for structural chromosome abnormalities has been attempted by a variety of approaches. The aims of PGD for translocations are to reduce the rate of spontaneous abortions that this population suffers and to minimize the risk of conceiving an unbalanced baby.
Mol Cell Endocrinol 2001 Oct 22
PMID:Preimplantation genetic diagnosis of structural abnormalities. 1157 34

Preimplantation genetic diagnosis (PGD) is an attractive addition to prenatal genetic diagnosis. Not only were traditional PGD indications valid, but newer indications should be envisioned. The major new indication is aneuploidy testing by PGD for transfer of euploid embryos. This could increase fertilization success in ART, and extend to couples experiencing repeated IVF failures or repeated spontaneous abortions. Other novel indications can be envisioned for the future.
Mol Cell Endocrinol 2001 Oct 22
PMID:Changing indications for preimplantation genetic diagnosis (PGD). 1157 37

The efficiency of animal production using cloning technology is still relatively low and research to determine a more efficient nuclear transfer procedure is ongoing. One approach which may be informative in assessing the viability of nuclear transfer embryos is the analysis of embryonic gene expression. Using RT-PCR techniques we have previously detected the aberrant expression of FGF4, FGFr2 and IL6 in a significant proportion of bovine granulosa cell-derived nuclear transfer embryos, which correlated with a limited developmental potential in vivo. In order to analyse the effect of different donor cell nuclei on embryonic gene expression we have now analysed the expression of these genes in nuclear transfer embryos reconstructed with fetal epithelial cell nuclei. In addition, we have compared the expression of these genes in bovine nuclear transfer embryos produced by cell fusion or direct injection with variations in the timing of oocyte activation. In all nuclear transfer embryos analysed, FGFr2 and IL6 transcripts were detected at a similar rate to that in IVF embryos. However, the absence of FGF4 transcripts was again evident in a large proportion of nuclear transfer embryos and most significantly in those embryos whose development was activated almost immediately following the transfer of the donor nucleus. The results demonstrate the effects that different donor cell lines and different nuclear transfer procedures may have on the expression of developmentally important genes in nuclear transfer embryos.
Mol Reprod Dev 2001 Nov
PMID:Comparison of gene transcription in cloned bovine embryos produced by different nuclear transfer techniques. 1159 38

Glucose is readily been taken up and utilized by preimplantation embryos from different species. However, a comprehensive analysis of the glucose transporter expression throughout preimplantation development is still missing. Here, we have investigated the expression of facilitative glucose transporters (Glut1-5 and 8) and sodium-dependent-glucose transporter (SGLT-I) in bovine oocytes and preimplantation embryos up to d16 of development, using RT-PCR and immunohistochemistry. The embryos were produced in vitro by IVM-IVF. Glut1, Glut3, Glut8, and SGLT-I were expressed in all stages studied. Glut4 transcripts were first detected at the blastocyst stage. Glut2 expression was restricted to the period of blastocyst elongation at d14 and d16. Transcription of the fructose transporter Glut5 started at the 8-/16-cell stage. Our results show a distinct expression pattern for glucose transporters during bovine embryo development in vitro indicating specialized functions for these isoforms at different developmental stages in bovine embryos. Mol. Reprod. Dev. 60:370-376,
Mol Reprod Dev 2001 Nov
PMID:Glucose transporter expression is developmentally regulated in in vitro derived bovine preimplantation embryos. 1159 48

Prolactin is mainly known for its role in breast development and lactation, but has been also implicated in other physiological functions such as immunoregulation and ovarian steroid production. Although prolactin and prolactin receptor (PRL-R) transcripts have been previously identified in the human ovary, the spatial localization of the receptor is unknown. To investigate the presence of PRL-R within the follicular apparatus, human luteinized granulosa cells were obtained at the time of follicular aspiration from women undergoing ovarian stimulation for IVF. RNA extracted from these cells was subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primers for the PRL-R gene. In addition, paraffin sections of isolated granulosa cells and sections of premenopausal human ovaries were immunostained with a mouse anti-human PRL-R monoclonal antibody. PRL-R were immunolocalized to the cell membrane of isolated luteinized granulosa cells and PRL-R transcripts were detected in the extracted RNA. No detectable staining was noted in secondary and early antral follicles in archived paraffin sections. These findings confirm the presence of PRL-R in human luteinized granulosa cells and suggest a localized role for PRL within the mature follicle. The absence of PRL-R in the early follicle suggests that the effects of prolactin are exerted around the time of ovulation.
Mol Hum Reprod 2001 Nov
PMID:Prolactin receptor gene expression and immunolocalization of the prolactin receptor in human luteinized granulosa cells. 1167 69

Butyrolactone I (BL-I) and Roscovitine (ROS), two specific and potent inhibitors of M-phase promoting factor (MPF) kinase activity, were used to block germinal vesicle breakdown (GVBD) of cattle oocytes. A concentration 6.25 microM BL-I and 12.5 microM ROS blocked over 93.3 +/- 2.5% of oocytes in germinal vesicle (GV) stage during a 24-hr culture period. Following a second 24-hr culture step in maturation medium (IVM) almost all (91.5 +/- 3.0%) inhibited oocytes resumed meiosis and reached the metaphase II (MII) stage. The MII kinetics was different for inhibited and control oocytes. Fifty percent MII was reached at 13-14 hr in BL-I + ROS treated oocytes, compared to 18 hr in control oocytes. Therefore, control oocytes were fertilised (IVF) after 22 hr IVM and inhibited oocytes after 16 or 22 hr IVM. After IVF, percentage of grade 1 freezable embryos on day 7 (D + 7) as well as percentage of blastocyst formation on D + 8 in the group of BL-I + ROS treated oocytes fertilised after 16 hr IVM were higher (P < 0.05) compared with the other experimental group fertilised after 22 hr IVM but not different in comparison with the control. Survival to freezing and thawing of grade 1 embryos frozen on D + 7 was employed as viability criteria and was similar in all groups. Thus, the presence of BL-I + ROS in the prematuration medium of bovine oocytes determines a reversible meiotic block, without compromising their subsequent developmental competence.
Mol Reprod Dev 2001 Dec
PMID:Bovine oocytes treated prior to in vitro maturation with a combination of butyrolactone I and roscovitine at low doses maintain a normal developmental capacity. 1174 69

The aim of this study is to examine the effect of bovine oocyte maturation, fertilization or culture in vivo or in vitro on the proportion of oocytes reaching the blastocyst stage, and on blastocyst quality as measured by survival following vitrification. In Experiment 1, 4 groups of oocytes were used: (1) immature oocytes from 2-6 mm follicles; (2) immature oocytes from > 6 mm follicles; (3) immature oocytes recovered in vivo just before the LH surge; and (4) in vivo matured oocytes. Significantly more blastocysts developed from oocytes matured in vivo than those recovered just before the LH surge or than oocytes from 2-6 mm follicles. Results from > 6 mm follicles were intermediate. All blastocysts had low survival following vitrification. In Experiment 2, in vivo matured oocytes were either (1) fertilized in vitro or (2) fertilized in vivo by artificial insemination and the resulting presumptive zygotes recovered on day 1. Both groups were then cultured in vitro. In vivo fertilized oocytes had a significantly higher blastocyst yield than those fertilized in vitro. Blastocyst quality was similar between the groups. Both groups had low survival following vitrification. In Experiment 3a, presumptive zygotes produced by in vitro maturation (IVM)/fertilization (IVF) were cultured either in vitro in synthetic oviduct fluid, or in vivo in the ewe oviduct. In Experiment 3b, in vivo matured/in vivo fertilized zygotes were either surgically recovered on day 1 and cultured in vitro in synthetic oviduct fluid, or were nonsurgically recovered on day 7. There was no difference in blastocyst yields between groups of zygotes originating from the same source (in vivo or in vitro fertilization) irrespective of whether culture took place in vivo or in vitro. However, there was a dramatic effect on blastocyst quality with those blastocysts produced following in vivo culture surviving vitrification at significantly higher rates than their in vitro cultured counterparts. Collectively, these results indicate that the intrinsic quality of the oocyte is the main factor affecting blastocyst yields, while the conditions of embryo culture have a crucial role in determining blastocyst quality.
Mol Reprod Dev 2002 Feb
PMID:Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. 1180 60

Although cryopreservation of bovine embryo has made great progress in recent years, little achievement was obtained in ovine embryo freezing, especially in vitro produced embryos. However, a simple and efficient method for cryopreservation of sheep embryos will be important for application of ovine embryonic techniques such as in vitro fertilization, transgenic, cloning and etc. In this study ovine blastocysts, produced in vivo or in vitro, were cryopreserved by vitrification in EFS40 (40% ethylene glycol (EG), 18% ficoll and 0.5 M sucrose) or GFS40 (40% glycerol (GL), 18% ficoll and 0.5 Mol sucrose). In vitro produced, early blastocysts were directly plunged into liquid nitrogen (LN2) after preparation by one of the following procedures at 25 degrees C: (A) equilibration in EFS40 for 1 min; (B) equilibration in EFS40 for 2 min; (C) equilibration in EFS40 for 30 s following pretreatment in 10% EG for 5 min; (D) equilibration in EFS40 for 30 s following pretreatment in EFS20 for 2 min (E) equilibration in GFS30 for 30 s following pretreatment in 10% GL for 5 min. The survival rates observed after thawing and in vitro culture for 12 h were A 78.0% (39/50), B 50.0% (26/52), C 93.3% (70/75), D 92.0% (46/50) and E 68.0% (34/50). Survival rates were not significantly different for treatments C and D (p>0.05), but those for groups C and D were significantly higher than for A, B and E (p<0.05). After 24 h in vitro culture, hatched blastocyst rates were A 28.0% (14/50), B 21.1% (11/52), C 49.3% (37/75), D 48.0% (24/50), E 32.0% (16/50) and control 54.0% (27/50). The hatching rates for groups A, B and E were significantly lower than the control (p<0.05) in which early IVF blastocysts were cultured in fresh SOFaaBSA medium following treatment in PBS containing 0.3% BSA for 30 min, but for groups C and D it was similar to the control (p>0.05). The freezing procedures A, B and C were used to vitrify in vivo produced, early blastocysts recovered from superovulated ewes. The survival rates of frozen-thawed in vivo embryos were A 94.7% (72/76), B 75.0% (45/60) and C 96.4% (54/56) and for group B was significantly lower than for the other two treatment groups (p<0.05). Hatched blastocyst rates were A 46.0% (35/76), B 26.6% (16/60), C 51.8% (29/56) and the control 56.7% (34/60) in which early blastocysts from superovulation were cultured in fresh SOFaaBSA medium following treatment in PBS containing 0.3% BSA for 30 min. The hatching rate for treatment B was significantly lower than for the control (p<0.05) but did not differ between groups A, C and the control (p>0.05). Frozen-thawed embryos vitrified by procedure C were transferred into synchronous recipient ewes. Pregnancy and lambing rates were similar for embryos transferred fresh or frozen/thawed for both in vivo and in vitro produced embryos. These rates did not differ between in vivo and in vitro embryos transferred fresh (p>0.05). However, for frozen-thawed embryos, both rates were significantly lower for in vitro than for in vivo produced embryos (p<0.05).
 ...
PMID:Vitrification of in vivo and in vitro produced ovine blastocysts. 1180 35

Insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) have been shown to be involved in ovarian follicular growth/development and steroidogenesis. Recently, a number of low-affinity IGFBP-related proteins (IGFBP-rP) have been characterized. In this study, we investigated the expression of the gene for IGFBP-rP2 (also known as connective tissue growth factor, CTGF) in human granulosa cells in vitro and in vivo. Northern blot analysis demonstrated that IGFBP-rP2 mRNA is expressed in cultured human granulosa-luteal cells obtained from women undergoing an IVF programme. Accumulation of IGFBP-rP2 mRNA was dose-dependently down-regulated by FSH and LH after 24 h treatment (both P < 0.05) in cultured granulosa-luteal cells. The inhibitory effects of gonadotrophins were mimicked by treatment with the protein kinase A activator, (Bu)(2)cAMP. Protein kinase C inhibitor staurosporine reduced, whereas protein kinase C activator TPA (12-O-tetradecanoyl phorbol 13-acetate) increased, IGFBP-rP2 mRNA accumulation. These results suggest that the inhibitory effects of gonadotrophins on IGFBP-rP2 gene expression may involve signal transduction via both protein kinase A and C pathways. Immunohistochemical analysis revealed positive staining for IGFBP-rP2 in the granulosa and theca cells of normal human ovarian follicles. Corpus luteum and ovarian surface epithelial cells were also positively stained. Modulation of IGFBP-rP2 expression by gonadotrophic hormones may have a role in ovarian follicular development and in the ovulatory process.
Mol Hum Reprod 2002 Feb
PMID:Gonadotrophins inhibit the expression of insulin-like growth factor binding protein-related protein-2 mRNA in cultured human granulosa-luteal cells. 1181 16


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