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PubMed2004

Detection of granulocyte colony-stimulating factor and its receptor in human follicular luteinized granulosa cells.

OBJECTIVE: To evaluate concentration of granulocyte colony-stimulating factor (G-CSF) in serum and follicular fluid (FF) at the time of oocyte retrieval and to detect expression of G-CSF and its receptor by luteinized granulosa cells (GCs). DESIGN: Collection of serum and FF at the time of oocyte retrieval. SETTING: A university IVF-ICSI program. PATIENT(S): Serum and FF were obtained from 82 women undergoing oocyte retrieval. INTERVENTION(S): Serum and FF were obtained from 82 women. Granulosa cells were pooled from 15 patients (three experiments with five patients each; 3-14 oocytes each). MAIN OUTCOME MEASURE(S): Granulocyte colony-stimulating factor concentration was determined by ELISA, the expression of G-CSF, and its receptor by the immunocytochemical technique and reverse transcriptase polymerase chain reaction analysis. Additionally, G-CSF expression was investigated by cell culture time course studies. RESULT(S): The median G-CSF level in FF (117.98 pg/mL) was significantly higher than that in serum (67.5 pg/mL). Granulocyte colony-stimulating factor and its receptor were expressed by GCs. CONCLUSION(S): The significantly higher level of G-CSF in FF than in serum and the expression of G-CSF and its receptor in FF by GCs suggest an important role for this growth factor in ovarian function.

Adult↗
PubMed2005

ICSI outcomes in obstructive azoospermia: influence of the origin of surgically retrieved spermatozoa and the cause of obstruction.

BACKGROUND: Spermatozoa can be retrieved from the testis and epididymis of men with obstructive azoospermia (OA) and used for ICSI. However, it is unknown whether the outcome of ICSI depends on the cause of obstruction or the origin of surgically retrieved spermatozoa. METHODS: A cohort of 171 men with OA and normal spermatogenesis were included in this retrospective study. They were divided into three groups according to the site and origin of obstruction: 83 men had congenital bilateral absence of vas deferens; 55 and 33 had acquired epididymal and deferent duct obstructions, respectively. The outcome of 368 ICSI cycles was determined and compared according to the origin of spermatozoa: epididymal (n = 253) or testicular (n = 115). RESULTS: Fertilization and clinical pregnancy rates did not differ between spermatozoa of different origin (58.9% versus 51.9% and 22.1% versus 24.3% with epididymal and testicular spermatozoa, respectively). However, the miscarriage rate was significantly higher for testicular spermatozoa (35.7% versus. 12.5% P < 0.05, chi2 test). Findings were similar whatever the aetiology of the OA. CONCLUSION: This study suggests that the use of testicular spermatozoa, even those generated during normal spermatogenesis, alters embryonic development and that epididymal spermatozoa should be preferentially used, irrespective of the aetiology of OA.

Adult↗
PubMed2005

Prolonged HCG action affects angiogenic substances and improves follicular maturation, oocyte quality and fertilization competence in patients with polycystic ovarian syndrome.

BACKGROUND: The aim of this study was to determine whether, in polycystic ovarian syndrome (PCOS) patients, HCG action prolonged for 4 h improves the action of angiogenic substances [ovarian renin angiotensin system and vascular endothelial growth factor (VEGF)], and consequently follicular maturation, oocyte quality and oocyte fertilization competence. METHODS: In this prospective study 20 patients with PCOS undergoing IVF were included. Oocyte retrieval was carried out either 34 or 38 h after HCG administration. Each follicle was analysed for prorenin, active renin, VEGF and estradiol. Oocytes were evaluated for quality (mature, immature, degenerated oocytes), as were the embryos (low or high). RESULTS: In the HCG +38 h group there were 245 follicles, and in the HCG +34 h group 240 follicles. In the HCG +38 h group, log active renin was lower (2.78 +/- 0.20 versus 2.91 +/- 0.25; P < 0.001) and VEGF higher (2276.0 +/- 790.1 versus 1946.6 +/- 954.5 pg/ml; P < 0.001). The odds ratio for obtaining oocytes from follicles was 1.6 [95% confidence interval (CI) 1.1-2.6; P = 0.02], and for developing high quality embryos 7.6 (95% CI 2.8-20.9; P < 0.001) in favour of the HCG +38 h group. CONCLUSIONS: Follicular maturation and oocyte quality are related to the intrafollicular influences of active renin and VEGF in a time-dependent manner after HCG administration, whereas fertilization competence is related to VEGF only.

Adult↗
PubMed2002

Pregnancies following blastocyst stage transfer in PGD cycles at risk for beta-thalassaemic haemoglobinopathies.

BACKGROUND: Preimplantation genetic diagnosis (PGD) usually involves blastomere biopsy 3 days post-insemination (p.i.), followed by genetic analysis and transfer of unaffected embryos later on day 3 or 4. We evaluate a strategy involving embryo biopsy on day 3 p.i., genetic analysis on day 4 and, following culture in blastocyst sequential media, transfer of unaffected embryos on day 5 p.i. METHODS: PGD cycles were initiated in 15 couples at risk of transmitting beta-thalassaemia major. Oocyte retrieval and ICSI were performed according to standard protocols. Embryo culture used blastocyst sequential media. Embryos were biopsied on day 3 p.i. using acid Tyrode's for zona drilling, and the single blastomeres were genotyped by a protocol involving nested polymerase chain reaction and denaturing gradient gel electrophoresis analysis. RESULTS: Forty of 109 (37%) embryos biopsied on day 3 p.i. developed to blastocysts by day 5 p.i., with at least one blastocyst available for transfer in 12 cycles (80%). Genotype analysis characterized 51/109 (47%) embryos unaffected for beta-thalassaemia major, of which 28 were blastocysts. Transfer of 37 day 5 p.i. embryos (blastocysts and non blastocysts) initiated eight clinical pregnancies. Implantation rate per embryo transferred was 12/37 (32%). CONCLUSIONS: Embryo biopsy on day 3, followed by delayed transfer until day 5 p.i. offers a novel and effective strategy to overcome the time limit encountered when performing PGD, without compromising embryo implantation.

Biopsy↗
PubMed2001

Gonadotropin-releasing hormone antagonist protocol: a novel method of ovarian stimulation in poor responders.

OBJECTIVE: To estimate the efficacy of gonadotropin-releasing hormone (GnRH) antagonist 'Cetrorelix' in poor responders comparing with the standard long protocol. DESIGN: The study population consisted of 21 poor responders who underwent ICSI and treated with Cetrorelix according to the multiple-dose protocol and who were compared with 21 poor responders treated according to the long protocol and who also underwent ICSI. Patients in both groups were matched for chronological age, the number of follicles found by ultrasound at the retrieval day and cause of infertility. Fifteen patients of GnRH antagonist group were treated with the combination of GnRH antagonist with clomiphene citrate (CC) plus gonadotropins, while six patients were treated with the combination of GnRH antagonist plus gonadotropins, but without CC. RESULTS: The use of GnRH antagonist in a multiple dose protocol gave a pregnancy rate of 14.28% which was in the range expected for patient with poor response, but with shorter treatment duration and with fewer ampoules of gonadotropins as compared with the use of a GnRH agonist protocol in a depot formulation. Within Cetrorelix group patients who received CC had a significant shorter duration of stimulation and needed fewer ampoules as compared with patients in the same group who did not receive CC. CONCLUSIONS: A GnRH antagonist multiple dose protocol may be the protocol of choice for the treatment of poor responders. The use of GnRH antagonist Cetrorelix ended with significantly less ampoules of gonadotropins and a shorter duration of stimulation.

Adult↗
PubMed2005

Factors influencing response to ovarian stimulation.

Ovarian stimulation is an integral part of assisted reproduction treatments. Ovarian response to gonadotrophin treatment, besides other factors, determines the outcome of treatment, as the number and quality of oocytes retrieved are related to the chance of achieving a pregnancy. A number of factors have been identified that might predict ovarian response, such as age of the patient and antral follicle count. In addition, it has been shown that genetic factors such as the patient's FSH-receptor genotype also determine individual response to FSH treatment. Besides patient-related factors, the choice of drugs for ovarian stimulation plays a significant role. Until recently, biopotency of gonadotrophin preparations was tested by an in-vivo bioassay with an intrinsic variability up to 20%. Due to a superior manufacturing technique, follitropin alpha can now be filled by mass. This allows assessment of FSH with a precise SE-HPLC assay and variability of the FSH content between production lots has now been estimated at 1.6%. Results of recent studies indicate that treatment with follitropin alpha filled by mass results in consistent ovarian response, fewer treatment days and fewer cancelled cycles. This is an important step towards further minimizing drug-related variability of ovarian response to FSH treatment.

Epidemiologic Factors↗
PubMed2002

Zona thinning with a noncontact diode laser in ICSI embryos from women of advanced age.

PURPOSE: The objective of this study was to determine if the zona thinning (ZT) technique improved the rates of implantation and clinical pregnancy for patients aged > or = 38 years submitted to an ICSI program. METHODS: A total of 100 patients submitted to ICSI and aged > or = 38 years were divided in a prospective and randomized manner into two groups: Group I - patients submitted to ZT (n = 50); a laser diode with 1.48 microm wavelength (Fertilaser) was used for the ZT procedure with 1-2 irradiations of 10 ms applied to four different positions on the zona pellucida (ZP) of each embryo to thin 60-90% of the ZP (each point with a 15-20 microm length of ZT). Group II - patients with no ZT (n = 50). In both groups, embryo transfer was performed on the second or third day. RESULTS: The age of Group I patients (39.8 +/- 1.3) did not differ (p = 0.67) from that of Group II patients (40 +/- 1.9). The number of oocytes retrieved at metaphase II from Group I (6.4 +/- 4.2) and Group II (6.8 +/- 5) was similar (p = 0.94). Normal fertilization rates and cleavage rates were similar (p = 0.78 and p = 0.63, respectively) for Group I (71.5 +/- 22% and 96.7 +/- 11%) and Group II (73.5 +/- 19.7% and 96 +/- 11%, respectively). The number of embryos transferred was similar (p = 0.53) for the two groups (Group I = 3.1 +/- 1.3; Group II = 2.9 +/- 1.1). The thickness of the ZP of Group I embryos (16.9 +/- 2.4 microm) did not differ (p = 0.97) from that of Group II embryos (16.9 +/- 2.3 microm). The rates of embryo implantation and clinical pregnancy per embryo transfer were similar (p = 0.67, p = 0.61) for Group I (7 and 16%, respectively) and for Group II (8.2 and 22%, respectively). CONCLUSIONS: These results suggest that ZT in the population aged > or = 38 years may have no impact on ICSI success rates. However, this conclusion is limited to a situation in which length of the laser ZT was < or = 20 microm and the laser was applied to four different positions.

Adult↗
PubMed2002

Endometrial hormone receptors and proliferation index in the periovulatory phase of stimulated embryo transfer cycles in comparison with natural cycles and relation to clinical pregnancy outcome.

OBJECTIVE: To investigate the endometrial steroid receptors and proliferation index in GnRH analogue/hMG-stimulated cycles in comparison with natural cycles and their relation to clinical pregnancy outcome. DESIGN: Prospective observational study. SETTING: Tertiary referral center. PATIENT(S): Twenty-seven stimulated patients with GnRH agonist and hMG. Twenty normo-ovulatory patients were the natural cycle controls. INTERVENTION(S): Endometrial aspiration biopsies: in stimulated cycles on the day of oocyte retrieval within the ET cycle (Day OPU) (n = 20) or 2 days later (Day OPU + 2) (n = 7); in natural cycles on the natural day of ovulation (Day NO) (n = 10) or on the day of ovulation + 2 (Day NO + 2) (n = 10). MAIN OUTCOME MEASURE(S): Comparison of endometrial maturation, estrogen (ER) and P receptor (PR), and proliferation index by immunohistochemistry in natural and stimulated cycles, correlation with pregnancy outcome in stimulated cycles. RESULT(S): Stimulated cycles Day OPU showed significantly advanced endometrial maturation compared to natural cycles Day NO; stromal ER and glandular and stromal PR staining was lower in stimulated than in natural cycles, but higher on Day OPU than on Day NO + 2; proliferation index was lower in all stimulated cycles. Steroid receptors and proliferation index in stimulated cycles were unrelated to clinical pregnancy occurrence. CONCLUSION(S): Compared to natural cycles, ovarian stimulation induced an imbalance in endometrial ER and PR and led to a profound antimitotic effect in the peri-ovulatory phase. These parameters were, however, not predictive of clinical pregnancy in cycles with ET.

Adult↗
PubMed1983

Sea urchin egg cortical granule exocytosis is followed by a burst of membrane retrieval via uptake into coated vesicles.

Using improved fixation procedures we have found that extensive endocytotic activity is turned on at fertilization in eggs of three species of sea urchins. Beginning after completion of cortical granule exocytosis and after exocytotic pits have completely smoothed over, the entire activated egg surface engages in a limited period of extensive removal of membrane via uptake into coated vesicles. This "burst phase" lasts about 3-5 min after which the number of invaginating coated vesicles decreases rapidly. At the end of this burst phase all the patches of cortical granule membranes have disappeared, and the egg surface is left uniformly covered by microvilli. For the remainder of the first cell cycle coated pits continue to form at a slower but steady rate. Endocytotic activity continues past the time of first cleavage. There is distinct overlap in onset and duration of the burst phase of endocytosis with the period of medium acidification during normal development. However, activation of eggs in choline sea water, which inhibits acid secretion, results in an endocytic burst whose timing and duration are similar to those in normal eggs. The endocytic burst is, therefore, independent of cytoplasmic alkalinization. These results suggest, in accord with the two-step model of egg activation (D. Epel, R. A. Steinhardt, and R. A. Humphreys, 1974; Dev. Biol. 40, 245-255; D. Epel, 1978, Curr. Top. Dev. Biol. 12, 185-246) that initiation of endocytosis is most likely a Ca2+-dependent event.

Animals↗
PubMed2006

Plasma levels of soluble vascular endothelial growth factor receptor-1 may determine the onset of early and late ovarian hyperstimulation syndrome.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a life-threatening condition associated with ovarian stimulation. Its pathophysiology is unknown and its treatment continues to be empirical. Early (E)- and late (L)-OHSS occur in women at risk, though not in all cases. Vascular endothelial growth factor (VEGF) is related to increased vascular permeability in OHSS. We analysed the dynamics of the VEGF system in E- and L-OHSS. METHODS: A prospective cohort of women undergoing IVF-ICSI treatment were divided into groups. E-OHSS: Nonpregnant patients classified as women not at risk (group 1) (n = 11) and patients at risk who did not (group 2) (n = 18) and did (group 3) (n = 8) develop severe OHSS. Blood was drawn on the day of ovum retrieval (day 0) and 3, 6, 10 and 14 days later. L-OHSS: Single pregnancies classified as women who did not (group 4) (n = 8) and did develop (group 5) (n = 4) OHSS. Single pregnancies after oocyte donation (OD) (n = 4) were compared with groups 4 and 5 (IVF-ICSI). Blood was obtained weekly (weeks 4-12). Total VEGF (VEFG-A), free (f)-VEGF and soluble VEGF receptor 1 (sVEGFR-1) in plasma and in serum alpha2-macroglobulin (M) were also measured. RESULTS: Group 3 showed significantly (P < 0.05) higher VEFG-A and f-VEGF than group 1 on day 6 because of lower sVEGFR-1 secretion. Similarly, group 5 had significantly (P < 0.05) more VEFG-A and f-VEGF and less sVEGFR-1 than group 4. Oocyte donation was associated with decreased sVEGFR-1 secretion, and alpha2M was not relevant in OHSS development. CONCLUSION: In E- and L-OHSS, the ability to secrete sVEGFR-1 and bind VEGF seems to be the determinant factor in OHSS. f-VEGF acts locally in the ovary.

Adult↗
PubMedDate not supplied

Cryopreservation of spermatozoa of black marlin, Makaira indica (Teleostei: Istiophoridae).

As a first step towards the development of a method for the cryopreservation of black marlin spermatozoa, this study investigated the effect of dimethylsulfoxide (DMSO) concentration and pellet size on post-thaw spermatozoal motility. Spermatozoa were recovered from the spermatic duct of testes retrieved post-mortem from four adult black marlin caught in the Coral Sea spawning grounds of Australia. Undiluted spermatozoa were stored on ice for 4 to 10 hours during transport to shore, then evaluated for motility after activation in seawater (1:10 v:v). Spermatozoa were prepared for cryopreservation in pellets by extension (1:3 v:v) in a defined fish Ringer's solution to give two final DMSO concentrations of 2.5% or 5.0%. Diluted spermatozoa were frozen directly on a dry ice block in pellet sizes of either 0.25 ml or 0.50 ml. Frozen pellets were thawed in a water bath at 40 degrees C for 60 seconds and assessed for post-thaw motility following activation in seawater. Spermatozoa recovered within 50 minutes of death and chilled on ice for 4 to 10 hours showed a mean (+/- SEM) motility immediately following activation of 91.6 +/- 7.9%. 50% of the spermatozoa remained motile for approximately 4 to 5 minutes. Following cryopreservation, mean motility declined significantly across all cryoprotectant and pellet size combinations (P < 0.001) but spermatozoa frozen in 2.5% DMSO showed higher motility than those frozen in 5.0% DMSO (P = 0.014). Pellet size had no effect on post-thaw motility (P = 0.179).

Animals↗
PubMed2001

Performing ICSI using an injection pipette with the smallest possible inner diameter and a long taper increases normal fertilization rate, decreases incidence of degeneration and tripronuclear zygotes, and enhances embryo development.

PURPOSE: To compare the efficacy of two types of injection pipette used for ICSI, one with a larger (5-7 microns) inner diameter and a shorter taper with that inner diameter, and another with the smallest (3-5 microns) possible inner diameter and a longer taper with that inner diameter. METHODS: Retrieved oocytes at metaphase II stage were injected using one of two types of injection pipette, in 33 and 94 cycles, respectively, in a total of 127 cycles in 108 patients. RESULTS: In comparison to the injection pipette with a larger (5-7 microns) inner diameter and a shorter taper with that inner diameter, the injection pipette with the smallest (3-5 microns) possible inner diameter and a longer taper with that inner diameter increased normal fertilization rate ((70 +/- 3.6)% vs. (86 +/- 2.2)%; P = .001; mean +/- SEM); decreased the incidence of degeneration ((14 +/- 2.4)% vs. (5 +/- 1.4)%; P = .001) and tripronuclear zygotes ((1.0 +/- 0.35)% vs. (0.1 +/- 0.21)%; P = .03); increased Day-2 diploid embryos ((69 +/- 3.7)% vs. (85 +/- 2.2)%; P = .001) and good-quality Day-2 diploid embryos ((67 +/- 4.0)% vs. (79 +/- 2.4)%; P = .03), all per injected oocyte; and increased the number of blastomeres per good-quality Day-2 diploid embryo ((3.0 +/- 0.21 vs. 3.8 +/- 0.12; P = .0003). CONCLUSIONS: Performing ICSI using an injection pipette with the smallest (3-5 microns) possible inner diameter and a longer taper with that inner diameter maximizes normal fertilization rate, minimizes the incidence of postinjection degeneration and tripronuclear zygotes, and enhances embryo development.

Adult↗
PubMed2001

Combined approach as an effective method in the prevention of severe ovarian hyperstimulation syndrome.

The purpose of this study was to evaluate the effectiveness of combined approach on the prevention of severe ovarian hyperstimulation syndrome (OHSS) in high risk patients undergoing controlled ovarian hyperstimulation for IVF. The combined approach consisted of: (1) step-down administration of gonadotropins; (2) lowering the dose of human chorionic gonadotropin; (3) intravenous albumin infusion at the time of oocyte retrieval and (4) progesterone use for luteal support. Total of 87 high risk patients with a serum estradiol level >11,010 pmol/l or 3000 pg/ml on HCG day were managed by this combined approach and their results were compared with 274 low risk patients. In all high risk patients, the gonadotrophin dose were decreased starting as early as on day 4 of ovarian stimulation as necessary, ovulation was triggered by a decreased HCG dose of 5000-7000 IU according to the level of estradiol, intravenous infusion of 20% human albumin, 50-100ml were given just 1h before the oocyte retrieval and luteal support was provided either by 50mg progesterone in oil, IM or 600 mg micronized progesterone orally or vaginally until the day of beta-HCG determination. All patients were followed by serial ultrasonographic examinations and complete blood count analysis after embryo transfer to detect the early signs of OHSS and to allow early intervention. Age and duration of infertility were similar in both groups. Although the number of gonadotrophin ampoules used (22.7 +/- 4.7 versus 27.8+/-3.7; P<0.05) was significantly lower, estradiol levels (16,764 +/- 6936 pmol/l versus 8870 +/- 2456 pmol/l; P<0.05) and mean number of oocytes (18.3 +/- 5.9 versus 10.6+/-5.4; P<0.05) were significantly higher in study group. There was no significant difference between groups in terms of the mean number of transferred embryos (3.2 +/- 1.1 versus 3.4+/-1.1) and rate of pregnancies (50.5% versus 40.1%). There was only one moderate and no severe OHSS case in the high risk group, while five moderate and one severe OHSS cases developed in the control group consisting of low risk patients. In conclusion, intravenous albumin combined with low dose HCG, early step-down administration of gonadotropins and progesterone use for luteal support, so called combined approach, proved to be effective in the prevention of severe ovarian hyperstimulation syndrome in documented high risk patients.

Adult↗
PubMed2005

Microtubule turnover in ooplasm biopsy reflects ageing phenomena in the parent oocyte.

Oviductal oocytes retrieved from superovulated B6D2F1 mice at 13.5, 16 and 19 h after human chorionic gonadotrophin (HCG) (groups A, B and C respectively, n = 382) were micromanipulated to obtain 12-20 mum sized ooplasm biopsy fragments. Experiments were divided into three sets. Ooplasmic microtubule dynamics were studied in ooplasm biopsy specimens and parent oocytes (set 1) and ooplasm biopsy specimens (set 2), whilst zona pellucida dissolution time, cortical granule loss and spindle/chromatin morphology using confocal microscopy were also studied in parent oocytes (set 2). Oocytes withstood oocyte biopsy with a high survival rate (98.2%) and the biopsied oocytes underwent successful fertilization and development (set 3). An absolute one-to-one correlation was seen between the oocyte biopsy specimens and the parent oocytes in terms of ooplasmic microtubule dynamics (set 1), and increased ooplasmic microtubule dynamics in oocyte biopsy specimens paralleled ageing phenomena in the parent oocytes (set 2). Zona pellucida dissolution time was significantly lower in parent oocytes from group A versus groups B (P = 0.032), and C (P < 0.001). (Groups A, B, C include minimal, moderate, increased ooplasmic microtubule dynamics in oocyte biopsy specimens respectively.) Oocyte cortical granule loss and spindle/chromatin abnormalities were mainly seen in group C (P < 0.001). Oocyte biopsy can thus be applied to judge age-related changes in the parent oocytes.

Animals↗
PubMed2002

ESHRE Preimplantation Genetic Diagnosis Consortium: data collection III (May 2001).

The ESHRE PGD Consortium was formed in 1997 to undertake a long-term study of the efficacy and clinical outcome of preimplantation genetic diagnosis (PGD). Here, the third report of the ESHRE PGD Consortium is presented, collating data received from 25 centres on referrals, cycles, pregnancies and babies born after PGD. The second report, published in December 2000, reported on 886 referrals, a total of 1318 started cycles (of which 465 for aneuploidy screening, 386 for fluorescence in-situ hybridization (FISH) and 385 for PCR going beyond oocyte retrieval), 163 pregnancies and 162 children born. This year, 675 referrals from 12 centres were added giving a total of 1561 referrals, 370 regular PGD cycles, 334 PGD-aneuploidy screening (PGD-AS) cycles and 78 cycles for social sexing from 24 centres and 215 pregnancies and 117 babies from 12 centres. Because more in-depth information was asked for the cycles, this year's data will be shown separately as well as cumulatively. One striking feature of this year's data collection is the appearance of the first data for gender screening on preimplantation embryos for social reasons. The ethical concerns regarding social sexing will be discussed, as well as the forthcoming changes in timing of data collection. When the data collection was discussed at the last meeting of the members of the ESHRE PGD Consortium in Lausanne, Switzerland (June, 2001), it appeared that the current system of data collection, although yielding results very quickly, showed fundamental flaws. The ESHRE PGD Consortium Steering Committee intends to remedy to these problems, on the one hand by introducing a new type and timing of data collection, and on the other hand by re-analysing and correcting the data which have already been sent in during the past 4 years.

Aneuploidy↗
PubMed2001

Morphology of in-vitro matured oocytes: impact on fertility potential and embryo quality.

BACKGROUND: The purpose of the present study was to investigate the morphology of in-vitro matured metaphase II (MII) oocytes and to observe if there was a difference in the morphology between polycystic and normal ovaries. Furthermore, the morphology of in-vitro matured MII oocytes was related to their subsequent fertilization and cleavage rates and to embryo quality. METHODS: This retrospective study included 264 MII oocytes obtained in 100 consecutive cycles. Oocyte retrieval was performed transvaginally and cumulus enclosed oocytes were matured for 28--30 h before evaluation. Prior to ICSI, all MII oocytes were graded into three groups according to the number of anomalies: grade I: oocytes without any anomaly (n = 144, 54%), grade II: oocytes with one anomaly (n = 87, 33%) and grade III: oocytes with at least two anomalies (n = 33, 12.5%). RESULTS: Oocyte grades did not differ between women with polycystic ovaries [grades I, II and III respectively: 58/94 (61.7%), 29/94 (30.9%) and 7/94 (7.4%)] and women with normal ovaries [grades I, II and III respectively: 86/170 (50.6%); 58/170 (34%); 26/170 (15.3%)]. Morphology was not related to fertilization rates. The cleavage rate was, however, affected by morphological anomalies (grade I [77/144 (53.5%) versus grade II 33/87 (37.9%) (P = 0.03)], although no significant decrease in cleavage rate could be demonstrated when all grade II and III oocytes were compared with normal oocytes. Significantly more embryos of good quality developed after grade I oocytes [54/144 (37.5%)] compared with those from grade II and grade III oocytes (22/120; P = 0.001). The presence of cytoplasmic abnormalities significantly decreased the cleavage rate (P = 0.04) and also the number of good quality embryos (P < 0.001). CONCLUSION: The in-vitro maturation of oocytes without anomalies yields higher quality embryos, with higher cleavage rates, than those with anomalies.

Cells, Cultured↗
PubMed2000

In vitro blastocyst formation of human oocytes obtained from unstimulated and stimulated cycles after vitrification at various maturational stages.

OBJECTIVE: To evaluate the developmental competence and chromosomal normality of oocytes vitrified at various times after maturation culture. DESIGN: In vitro model study. SETTING: A university-affiliated hospital. PATIENT(S): Unstimulated women who underwent cesarean section or oophorectomy and infertile women who underwent a long protocol of GnRH stimulation. INTERVENTION(S): Retrieved oocytes were vitrified at 0 or 48 hours after culture in unstimulated cycles and at 0, 8-15, or 24-28 hours after culture in stimulated cycles. MAIN OUTCOME MEASURE(S): Postthaw morphologic normality, maturation, fertilization, cleavage, blastocyst formation, and chromosome number. RESULT(S): In the 53 oocytes that were obtained from unstimulated cycles, no statistically significant differences were found in rates of morphologic normality (range, 56%-63%) or fertilization (range, 31%-37%) according to the time of vitrification. In the 50 oocytes that were obtained from stimulated cycles, more of those that were vitrified at 24-28 hours were morphologically normal than those that were vitrified at 0 or 8-15 hours. Regardless of these differences, high cleavage rates (83%-100%) were obtained that did not differ significantly among the treatment groups. In both cycles, 20%-43% of cleaved oocytes developed to the blastocyst stage by 6 days after IVF. All the karyotyped blastocysts, three from unstimulated cycles and four from stimulated cycles, had a normal number of chromosomes. CONCLUSION(S): Vitrified and thawed oocytes from unstimulated or stimulated cycles developed to the blastocyst stage, regardless of when vitrification occurred; the number of chromosomes in the blastocysts was normal.

Adult↗
PubMed1998

Birth of live mice derived by in vitro fertilization with spermatozoa retrieved up to twenty-four hours after death.

The objective of this study was to determine the viability and fertility of mouse spermatozoa obtained at various postmortem intervals. Male mice were euthanized, and the bodies were kept at approximately 22 degrees C for up to 24 hr. The epididymides were removed and spermatozoa were allowed to swim out into Minimum Essential Medium supplemented with bovine serum albumin. Motility of spermatozoa retrieved within 12 hr postmortem was approximately 60%, whereas motility of those obtained 6 to 12 hr later decreased significantly (P < 0.05). There appeared to be no differences in the percentages of spermatozoa with intact plasma and acrosomal membranes regardless of the time after death. After in vitro fertilization of oocytes with spermatozoa collected immediately after death or at 6, 12, 18, or 24 hr postmortem, the cleavage rates were 81%, 70%, 64%, 34%, and 19%, respectively. Once oocytes were fertilized, more than 65% developed into morulae/blastocysts. Transfer of a total of 166 embryos produced in vitro with postmortem spermatozoa resulted in the birth of 44 live pups (26.5%). Of these 44, 3 live mice were derived by transfer of 11 embryos (27.3%) produced with 24-hr postmortem spermatozoa. Histological examination of the testes and epididymides obtained at various postmortem intervals revealed that degenerative changes of the testes occurred within 6 hr, whereas those of the epididymides were less obvious until 6 hr later. These changes included pyknosis, release of intracellular contents, and disruption of intercellular bridges of the germ cells. This study has demonstrated that spermatozoa recovered from a dead animal as long as 24 hr after death can be used to fertilize oocytes, and that the resulting zygotes can develop into live young.

Acrosome↗