Efficacy of oocytes donated by older women in an oocyte donation programme

Population and insemination studies indicate that women experiencedeclining fertility with ageing. The question therefore ariseswhether older women are suitable oocyte donors. This study addressesthis issue by examining the relationship between oocyte donorage and clinical outcome in a large oocyte donation programme.We retrospectively reviewed data from 458 consecutive oocytedonation cycles completed by 164 different designated oocytedonors. Data were divided into two groups: group A, cycles withdonors aged 21–30 years at the time of follicular aspiration(193 cycles, 88 donors); and group B, cycles with donors aged31–40 years at the time of follicular aspiration (265cycles, 86 donors). Five donors, because of ageing during repetitivedonations, contributed data to groups A and B. In a given cycle,all oocytes for a recipient came from only one designated donor.Comparing the two donor groups, there was no difference in theamount of gonadotrophin used to achieve optimal stimulation;however, more oocytes were obtained from group A than groupB donors (16.8 ± 6.9 and 15.1 ± 8.1 respectively,P < 0.05). Similar percentages of oocytes were fertilizedin each group, resulting in the transfer of comparable numbersof embryos (4.5 ± 1.1 and 4.4 ± 13 respectively).Comparable clinical pregnancy rates were achieved (group A,36%; group B, 37%). The spontaneous abortion rates were alsosimilar (group A, 20%; group B, 12%), resulting in comparableongoing and delivered pregnancy rates per cycle (group A, 29%;group B, 32%) and per embryo transferred (group A, 6.4%; groupB, 7.3%). In conclusion, women of proven fertility should notbe excluded from donating oocytes simply because of their age.There exists a cohort of fertile women who resist the decreasingfecundity and increasing spontaneous abortion rates associatedwith ageing. With careful screening, many women of proven fertilitycan donate oocytes until the age of 40 years with an efficacyequal to that of younger women. Given the relative shortageof suitable oocyte donors, and increasing requests from recipientswith previous donor oocyte babies to obtain oocytes from thesame, now older, donor, the findings of this study are of practicalclinical importance.     

An evaluation of semen analysis and in-vitro tests of sperm function in the prediction of the outcome of intrauterine AIH

Twenty-nine couples with an average of 5 years of infertilitywere selected for treatment by intrauterine insemination ofwashed semen (AIH). The criteria for selection were (i) thefemale partner showed no detectable fertility disorders by routinescreening; (ii) the male partner showed subnormal semen qualityon conventional semen analysis. Ovulation was stimulated uniformlywith clomiphene citrate and precipitated with human chorionicgonadotrophin (HCG). Inseminations were performed 31–32h post-HCG, with the day of HCG determined by ultrasound monitoringof follicular development. The fertilizing capacity of the malepartners‘ spermatozoa was tested in vitro using donatedhuman oocytes and/or the zona-free hamster oocyte penetrationassay. Up to eight cycles of AIH were alternated with cyclesof natural intercourse. While no pregnancies occurred in thegroup during normal coital cycles, the AIH pregnancy rate was17% per couple, but only 3% per insemination cycle. Four furtherpregnancies were achieved spontaneously in couples from thestudy group within 3 years of completion of the AIH therapyand four patients became pregnant following subsequent GIFTor IVF treatments. Neither of the in-vitro tests was helpfulin predicting the outcome of AIH, spontaneous pregnancy norof subsequent assisted conception procedures.     

Influence of sperm immobilization on onset of Ca(2+) oscillations after ICSI

Sperm immobilization prior to intracytoplasmic sperm injection (ICSI) is thought to be necessary for efficient fertilization. A variety of methods of sperm immobilization (pipetting, squeezing and piezo application) are currently employed in ICSI. The effect of differences in immobilization method on the timing of initial Ca(2+) oscillations of oocytes in ICSI was investigated. Motile spermatozoa were immobilized in eosin Y solution using pipetting, squeezing and piezo application. Complete staining of the sperm head was achieved after 220.7, 42.2 and 5.0 s respectively. Oscillations after ICSI were measured fluorometrically for each method. The onset of Ca(2+) oscillations was observed at 4.8 to 80.4 min after ICSI. Ca(2+) oscillations developed earlier with the piezo method (14.4 +/- 6.4 min) than other methods (pipetting, 43.1 +/- 20.2 min, P < 0.01; squeezing, 18.4 +/- 3.8 min, P = NS). The piezo method produced the earliest staining of the sperm head and may have caused the most damage to the sperm membrane. A more rapid onset of Ca(2+) oscillations was also observed with the piezo method. The method of sperm immobilization may be important for the rapid release of sperm factors that initiate oocyte activation. This study also showed that Ca(2+) oscillations develop earlier in human oocytes treated by ICSI than indicated in previous reports.     

Fertilization and early embryology: Effects of embryo density and co-culture of unfertilized oocytes on embryonic development of in-vitro fertilized mouse embryos

We have evaluated the effects of embryo density and the co-cultureof unfertilized (degenerating) oocytes on the development ofin-vitro fertilized (IVF) mouse embryos. In experiment 1, groupsof one, five, 10 or 20 zygotes were cultured in 20 µldrops of modified human tubal fluid (HTF) medium for 168 h at38.7°C in 5% CO₂ and 95% air. As the embryo density increased,significantly (P < 0.05) higher rates of embryos reachedhatched blastocyst stage. In addition, the time required forhatching after IVF was significantly (P < 0.05) shortenedby the increase in embryo density. In experiment 2, 10 IVF zygoteswere cultured with or without 10 unfertilized (degenerating)oocytes in 20 µl drops of HTF medium. The rates of IVFembryos that developed to morula, blastocyst, expanded blastocystand hatched blastocyst stages were decreased significantly (P< 0.01) by culturing embryos with unfertilized oocytes comparedwith culturing embryos alone. In experiment 3, groups of oneor 10 IVF zygotes or 10 IVF zygotes plus 10 unfertilized oocyteswere cultured in 20 µl drops of HTF medium and the numberof cells per blastocyst was examined at 120 h after IVF. Increasingembryo density resulted in a significant (P < 0.05) increasein the number of cells per blastocyst. In contrast, the cellnumber of IVF embryos that developed to blastocyst decreasedsignificantly (P < 0.05) when they were cultured with unfertilizedoocytes. The results suggest that in-vitro development of IVFmouse embryos is enhanced by increasing embryo density and isimpaired by co-culture with unfertilized (degenerating) oocytes.     

Follicular hyperplasia presenting with a marginal zone pattern in a reactive lymph node lesion

Histologically, the marginal zone pattern of the lymph node is characterized by lymphoid follicles with three distinct layers. The inner layer is composed of follicular center zones, the middle layer of darkly stained mantle zones, and the outer layer of marginal zones. However, the marginal zone pattern is rarely seen in reactive lymph nodes except for mesenteric lymph nodes. We describe the clinicopathologic, immunohistochemical and genotypic findings of six cases of reactive follicular hyperplasia exhibiting the marginal zone pattern. The patients comprised three males and three females (age range 24 to 63 years; medium 56 years). Follow-up data were obtained from five patients. None of them developed malignant lymphomas during the follow-up period of from 5 to 204 months (median 68 months). Histologically, the lesion was characterized by numerous lymphoid follicles and partial distortion of lymph node structure. Varying degrees of progressive transformation of the germinal center (PTGC) were noted in the four cases. The marginal zone pattern was observed in some or most of the lymphoid follicles including PTGC. The marginal zone B cells were small to medium-sized lymphocytes with round or slightly indented nuclei and a broad rim of pale cytoplasm. Some of them had a monocytoid appearance. They were CD20+, CD79a+, sIgM+/-, sIgD-, CD5-, CD10-, CD21-, CD23-, CD43-, CD45RO-, Bcl-6-, cyclin D1-, EMA- and p53-. A portion of them were Bcl-2 positive. Occasional large lymphoid cells with round or indented nuclei and moderate amounts of cytoplasm were observed in the marginal zone in four cases. These large lymphoid cells were usually CD20 positive, but Bcl-6 negative. A small number of them contained polytypic intracytoplasmic immunoglobulins. The polytypic nature of B lymphocytes was demonstrated by immunohistochemistry and polymerase chain reaction. Recognition of unusual marginal zone hyperplasia in reactive lymph node lesions is important to avoid confusion with nodal involvement in various low-grade B cell lymphomas presenting a marginal zone distribution pattern.     

Integrins are not involved in the process of human sperm-oolemmal fusion

BACKGROUND: We investigated whether integrins are required forthe human sperm–oocyte binding and fusion processes. METHODS:The expression of several integrin subunits at the human oocyteplasma membrane was investigated using immunofluorescence microscopy,and the functional role of integrins expressed at the humanoocyte surface in sperm–oocyte interaction was studiedusing a zona-free human oocyte binding and fusion assay. A totalof 144 unfertilized oocytes were stained with anti-integrinantibodies and 147 zona-free unfertilized oocytes were inseminatedin the presence of various anti-integrin antibodies that wereexpressed in oocyte plasma membrane. RESULTS: The antibodiesof six integrin subunits (₂, ₃, ₅, ₆, _V, _M) and six integrinsubunits (₁, ₂, ₃, ₄, ₅, ₆) were bound to the surface of fixedunfertilized oocytes. In contrast, the presence of ₁ and ₄ subunitscould not be verified. The human sperm–oocyte bindingwas only partially inhibited by blocking antibodies of ₂, ₃,₅, ₆, _V, _M, ₁, ₂ and ₃ with a maximum of 55% inhibition, butantibodies of ₄, ₅ and ₆ showed no effect on sperm–oolemmalbinding. A similar reduction of the number of fused sperm wasobserved. However, the ratio of fused sperm to total sperm (boundand fused) was not impaired by all integrin antibodies, suggestingthat integrins had no role in the sperm–oolemmal fusionprocess. CONCLUSIONS: These results suggest that one of thebinding mechanisms can be inhibited by integrin antibodies butthat this mechanism does not play an essential role in the humansperm–oolemmal binding and fusion processes. The othermechanisms, insensitive to integrins, may involve both bindingand fusion processes in human oocytes.     

CA 125 in seminal plasma: correlation with semen parameters

Ovarian cancer marker CA 125 was measured in human seminal plasma,and the concentrations ranged between 22 and 1149 U/ml, andbetween 39 and 4711 U/ejaculate. This very high patient-to-patientvariability was in contrast to a much lower within-patient variability,which was comparable to that of other semen parameters. No significantdifferences in CA 125 concentration were found in seminal plasmafrom normospermic patients, patients with male factors, vasectomizedmen, and in aliquots of samples which led to a pregnancy, viaartificial insemination or in-vitro fertilization. The seminalplasma CA 125 concentration was not correlated with sperm count,motility and morphology. In contrast, seminal plasma CA 125concentrations correlated with the age of the patient (P <0.001) and inversely with the volume of the ejaculate (P <0.001). These correlations were independent of each other. CA125 did not correlate with the prostatic marker zinc, but diddo so with the seminal vesicle marker fructose and the epididymalmarker carnitine.     

A report on 100 cycles of oocyte donation; factors affecting the outcome

Eighty-two patients had 100 cycles of oocyte donation from 68 donors resulting in 27 clinical pregnancies. The source of donated oocytes was 42 fertile volunteers and 26 patients from the assisted conception programme. The pregnancy rate was significantly higher when intra-Fallopian transfer was performed (36%; 21/59), compared with intrauterine transfer (15%; 6/41). The pregnancy rate following fresh gamete/embryo transfer (39%; 15/39) was slightly higher than for frozen embryo transfer (20%; 12/61). The age of the recipient significantly affected the pregnancy rate. The pregnancy rate was 50% in the 25-29 years age group and steadily dropped to 9.7% in the 45-49 age group. The pregnancy rate in patients with primary ovarian failure (50%; 8/16) was significantly higher than in patients with secondary ovarian failure (18%; 9/50). The pregnancy rate was significantly greater when parous donors (33%; 23/69) were used compared with non-parous donors (13%; 4/31). The number of gametes/embryos transferred significantly affected the pregnancy rate regardless of the treatment used. If one or two gametes/embryos were transferred, the pregnancy rate was 11% compared with 33% if three to four embryos were transferred. The age of the donors did not affect the pregnancy rate. The majority of the donors were under the age of 35 years. The best results (50% per cycle) were therefore achieved using gametes from parous donors and transferring three to four oocytes fresh to the Fallopian tubes of a young recipient.     

Laser-assisted zona pellucida thinning prior to routine ICSI

BACKGROUND: In MII oocytes showing difficult oolemma breakage, ICSI can cause an increase in the degeneration rate. This may be overcome by laser-assisted ICSI using a 5-10 micro m opening in the zona pellucida for injection. However, such a small opening might impair the hatching process, especially if assisted hatching is applied in addition. In order to prevent this, the present study used routine injection through an area of zona pellucida in which laser zona thinning had been applied, providing for both a reduced mechanical stress to the oocyte and assisted hatching. METHODS: This prospective study involved 100 cycles with 1016 MII oocytes. Conventional ICSI (control group) was compared with a modified laser-assisted ICSI (study group) in sibling oocytes. In the latter group oocytes were injected through an extended area of zona thinning. RESULTS: Degeneration rate was significantly lower in the study group (P < 0.004). There were no differences in fertilization, or formation and quality of blastocysts. In the study group embryo quality on day 2 was significantly better (P = 0.004) and herniation of day 5 blastocysts was increased (P = 0.005). Rates of implantation and pregnancy were not affected. However, on day 3 laser-assisted ICSI proved beneficial (P = 0.038) in terms of clinical pregnancy rate. CONCLUSIONS: The new method combines a less invasive ICSI technique with assisted hatching. Our preliminary data indicate that in addition to an improved oocyte survival, this new approach increases the hatching rate in vitro, which may explain the increase in pregnancy rate, at least in day 3 transfers.     

Follicular fluid renin concentration and IVF outcome

Total renin protein concentration (TRC) was measured in stored follicular fluid (FF) samples from 42 women. Samples were selected according to their origin from follicles either without recovered ova ('empty', n = 38) or fertilized but with failed implantation ('failed', n = 36) or successful deliveries ('deliveries', n = 71). Ratios of number of embryos transferred to number of infants delivered were 2:1, 3:1 or 4:2 but 1:1 was not available. Non-parametric testing was applied to FF-TRC, volume and outcome. TRC was significantly higher in the delivery than the failed (P = 0.001) or empty (P = 0.002) categories. Assuming that the range of renin in failed follicles can identify the sub-population of unsuccessful follicles in the delivery category, then elevated FF-TRC was clearly associated with successful outcome. For individual women, the odds of infant delivery increased 17-fold as a function of average FF-TRC between 10,000 and 25,000 microIU/ml. For failed and delivery but not empty follicles, higher renin levels occurred in the smaller follicles, consistent with a burst of renin synthesis associated with the presence of an oocyte. The results suggest that FF-TRC relates to ovum viability with ovarian hyperstimulation and may have predictive use in IVF programmes.