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Increased fertilization and pregnancy rate in polypronuclear fertilization cycles in in vitro fertilization-embryo transfer.

OBJECTIVE: To compare fertilization and pregnancy rates between cycles with polypronuclear fertilizations and cycles with normal fertilizations. DESIGN: In vitro fertilization-embryo transfer (IVF-ET) cycles in which oocytes were retrieved were divided into two groups according to the nature of fertilization. PATIENTS: All patients were participants of our IVF-ET program. RESULTS: A significantly higher fertilization rate was found in the polypronuclear fertilization cycles (61% versus 36.6%) and also an improved pregnancy rate (47.5% versus 19.6%) and per embryo transfers (53% versus 28.8%). The vast majority of polypronuclear fertilizations occurred in mature oocytes. CONCLUSION: We believe that the increased receptability of the oocytes improves fertilization and conception rates. The polypronuclear fertilization is an extreme expression of such improved receptibility and should be considered as an encouraging sign for conception.

Embryo Transfer

Sperm creatine phosphokinase M-isoform ratios and fertilizing potential of men: a blinded study of 84 couples treated with in vitro fertilization.

OBJECTIVE: To examine the value of sperm creatine phosphokinase M-isoform (CK-MM) measurements toward predicting fertilizing potential of men. DESIGN: In 84 in vitro fertilization (IVF) couples without knowing the semen parameters, reproductive history or the outcome of the IVF cycles, we determined the sperm CK-MM ratios (the proportion of sperm CK-MM versus CK-MM+CK-BB). Husbands with less than 10% or greater than or equal to 10% CK-MM ratios were classified as "low likelihood for fertilization" (CKMM-Infertile, n = 22) or "high likelihood for fertilization" (CKMM-Fertile, n = 62), respectively. RESULTS: Both the CKMM-Infertile and CKMM-Fertile groups (CK-MM ratios: 4.9% +/- 0.6% versus 31.1% +/- 1.8%) were in the normospermic range (31.5 +/- 6.9 versus 78.4 +/- 5.9 x 10(6) sperm/mL and 45.6% +/- 5.0% versus 54.0% +/- 2.0% motility). The fertilization rates (6.2 versus 4.9 oocytes inseminated) were 14.2% versus 53.4%, and 72.7% versus 25.8% of the couples failed to achieve any oocyte fertilization. All 14 pregnancies (16.7% rate) occurred in the CKMM-Fertile group. The pregnancy rate in the 62 CKMM-Fertile couples was 22.6%, and considering only the 46 CKMM-Fertile women in whom oocyte fertilization occurred, it was 30.4%. Among the 22 CKMM-Infertile men, 9 were normospermic and 9 of the 62 CKMM-Fertile men were oligospermic. Within the CKMM-Fertile group, 12 and 2 of the 14 pregnancies occurred by the 53 normospermic and 9 oligospermic men (22.6% versus 22.2% rate). CONCLUSIONS: Sperm CK-MM ratios, a measure of normal sperm development, predict fertilizing potential independently from sperm concentrations. Sperm CK-MM ratios also detect unexplained male infertility (infertile men with normospermic semen), a diagnosis that until now could not be substantiated.

Biomarkers

Total acrosin activity correlates with fertility potential after fertilization in vitro.

OBJECTIVE: To evaluate the possible relationship between total acrosin activity in spermatozoa and fertility potential after fertilization in vitro. DESIGN: Total acrosin activity of spermatozoa was measured in 101 in vitro fertilization (IVF) cases by an observer unaware of fertilization and cleavage results. SETTING: University Hospital is a tertiary referral center offering a government supported In Vitro Fertilization Programme. PARTICIPANTS: Participants were couples undergoing IVF. INTERVENTIONS: A miniature assay measured total acrosin activity in the semen sample used for IVF. OUTCOME MEASURES: The proportion of mature oocytes fertilized, the proportion of mature oocytes transferred, and fertilization of at least one mature oocyte were considered outcomes with fertility potential. RESULTS: Total acrosin activity correlated with both the proportion of mature oocytes fertilized and the proportion of mature oocytes that were transferred as cleaving embryos. Total acrosin activity was higher in cycles when one or more mature oocyte fertilized compared with cycles with failed fertilization of all mature oocytes. CONCLUSIONS: The likelihood ratio for subnormal results indicates that measurement of total acrosin activity is a fair test of the fertilizing capacity of sperm.

Acrosin

Migration sedimentation technique as a predictive test for the fertilizing capacity of spermatozoa in an in-vitro fertilization programme.

The migration-sedimentation technique (MST) has been proposed as a means of separating high quality motile spermatozoa. The present study was conducted in order to evaluate whether sperm performance following separation by MST predicts their fertilizing capacity in an in-vitro fertilization (IVF) programme. Ninety semen specimens were analysed for use in an IVF-embryo transfer (ET) programme. Each specimens was divided into two parts: one was processed in the IVF programme and was used after sperm swim-up separation for insemination of human ova. The other aliquot (0.2 ml) was separated by MST, and the sperm then characterized by their concentration, motility, degree of motility and morphology. Sperm characteristics after separation by MST were then correlated with the results of the IVF-fertilization rates. In 79 of 90 IVF-ET cycles, at least one oocyte was fertilized. All post-MST sperm characteristics were significantly higher in cycles with fertilizations compared to IVF cycles without fertilization. A larger percentage of the total motile spermatozoa were recovered after MST in semen specimens with fertilization, compared to semen specimens without fertilization (39.9 +/- 3.6 and 20.6 +/- 6.6%, respectively; P < 0.05). This value was correlated with the percentage of fertilized oocytes (r = 0.24; P < 0.02). More IVF cycles with fertilizations were recorded in cases in which the recovery of motile sperm was > 25% (P < 0.005), or when more than 1.5 x 10(6) motile spermatozoa were recovered after MST (P < 0.0001). As sperm characteristics after MST correlated significantly with their fertilizing capacity, the MST test could be used in evaluation of the fertilizing capacity of spermatozoa.

Cell Separation

The relationship of semen parameters to fertilization in patients participating in a program of in vitro fertilization.

Approximately 80% of the patients in a program of in vitro fertilization (IVF) will fertilize an oocyte. The purpose of this study was to determine which parameters of the semen analysis influence fertilization in vitro. Of 120 patients participating in an in vitro fertilization program, 98 achieved fertilization of at least one mature oocyte and 22 did not. Ovulation induction was standardized and patients whose sperm was exposed to at least one mature oocyte (by light microscopy) were included in the study. Semen washing was accomplished using a "swim-up" technique. Semen parameters were assessed both before (raw) and after washing. Following insemination with 100,000 motile sperm, fertilization was determined by the presence of pronuclei or cleavage. Mean sperm count and motility were higher in patients who fertilized. However, morphology was similar. Fertilization was more likely to occur with a raw density greater than 104 million/ml and a motility greater than 64%, as well as with a density greater than 18 million/ml and a motility greater than 86% following washing. Furthermore, washing lowered sperm counts by 75% and increased motility by 25% but had no effect on morphology. This study demonstrates that sperm count and motility, but not morphology, influence fertilization in a program of in vitro fertilization and that patients with higher counts and motility have a greater probability of fertilization.

Female

Fertility of cooled and frozen rabbit sperm measured by competitive fertilization.

The fertility of rabbit sperm that had been cooled to 5 degrees C or frozen and thawed was determined by competitive fertilization. Treatments were identified by labeling sperm with fluorescein isothiocyanate (FITC) or tetramethylrhodamine B isothiocyanate (TRITC). Sperm from different treatments were mixed and used in a competitive insemination experiment. Does were inseminated 5, 10 or 15 h prior to ovulation. Time of ovulation was controlled by injections of luteinizing hormone. The functional sperm transport, as determined by the number of sperm transported to the site of fertilization and capable of fertilizing oocytes, was estimated by counting the total number of differently stained sperm that surrounded or fertilized each oocyte. The fertility of sperm cooled to 5 degrees C was not affected (p less than 0.05) as compared to fertility of uncooled sperm. Functional sperm transport at all times of insemination and fertilization ratio at insemination 10 or 15 h before ovulation were reduced (p less than 0.05) for frozen-thawed vs. cooled sperm. No difference in fertilization ratio (p greater than 0.05) occurred, however, when does were inseminated 5 h before ovulation. While sperm survival and capacitation time appeared to play roles in fertility of frozen-thawed sperm, the most important factor was reduced functional sperm transport. However, fertility of frozen-thawed sperm was improved when the time from insemination to ovulation was reduced.

Animals

The use of rat in vitro fertilization to detect reductions in the fertility of spermatozoa from males exposed to ethylene glycol monomethyl ether.

An in vitro fertilization (IVF) assay sensitive enough to detect changes in the fertilizing capacity of spermatozoa would be a useful tool with which to investigate the action of testicular toxicants. A known testicular toxicant, ethylene glycol monomethyl ether (EGME), was used to induce specific lesions in the germinal epithelium so that the ability of a rat IVF system to detect changes in fertility could be tested. Male rats were given single, oral doses of 50, 100, and 200 mg EGME/kg. Spermatozoa were recovered from the cauda epididymides of these males at intervals after treatment; their fertility was assessed using IVF, and the testes were processed for histologic examination. The fertility of the control males was consistently greater than 65%. Spermatozoa from males treated with EGME had reduced fertility at specific times after dosing. Thus, after 50 mg EGME/kg there was reduced fertility at 5 weeks; after 100 mg EGME/kg there was reduced fertility at 3.5, 4.5, 5, 6, and 6.5 weeks, and after 200 mg EGME/kg there was reduced fertility at 2 and 3 weeks, between 4.5 and 6 weeks, and at 7 weeks. This corresponded to damage to the elongated spermatids (2, 3, and 3.5 weeks), pachytene spermatocytes (4.5 to 6 weeks), and leptotene and preleptotene spermatocytes (7 weeks). This accords well with the data from serial breeding trials and reports of histologic damage after exposure to EGME. Therefore, using IVF it was possible to detect EGME-induced changes in fertilizing capacity which correlated closely with observations of testicular damage. It was also possible to demonstrate a clear dose response to EGME.

Animals

Relationship of bull fertility with daughter fertility and production traits in Holstein dairy cattle.

The phenotypic and genetic correlations between fertility ratings of AI bulls for conception rate and their estimated breeding values for daughters' fertility and production traits were calculated. Genetic correlations between fertility ratings of bulls for conception and heifer fertility traits (age at first breeding, age at last breeding, and number of insemination per conception) were negative and ranged from -.04 to -.23, indicating daughters of bulls with high fertility ratings were younger at first breeding and required fewer services to conceive. In general, genetic correlations between fertility ratings of bulls for conception rate and cow fertility traits (days from calving to first breeding, days open, and number of inseminations per conception) and production traits (breed class average milk and fat and fat percentage) in the first two lactations were also moderate to high and in the favorable direction. Although heritability of both male and female fertility is low, these data indicate that heavy use of sires with high fertility ratings could have a mild positive effect on both male and female fertility. Evidence is also found to indicate that in this breed, selection for increased milk yield should not impair genetic ability of cows to reproduce.

Animals

[Problems in evaluating male fertility: valuable factors in evaluating male fertility and normal values of seminal parameters].

To determine the valuable factor for evaluating male fertility, a comparative study was done as to various seminal parameters between fertile and infertile groups. The fertile group consists of 57 proven fertile males and the infertile group consists of randomly chosen 67 infertile patients. Seminal parameters assessed were sperm concentration, motility, mean velocity, total sperm output, total motile sperm output, sperm morphology, acrosin activity and sperm penetration rate on zona-free hamster egg penetration assay (SPA). The infertile group was significantly different from the fertile group in every parameter except acrosin activity. However, the range of each parameter in the two groups overlapped each other. The diagnostic rate of each parameter, which is the percentage of an infertile male correctly diagnosed as infertile, was calculated by using 95% specificity threshold value of fertile males. The 95% specificity threshold values of sperm concentration, motility and % normal shaped sperm were 24.9 x 10(6)/ml, 34.9% and 55%, respectively, and they could be acceptable for the normal limit of seminal parameters. The diagnostic rate was highest in penetration rate (72.4%). In other words, penetration rate is the most valuable factor in various parameters for making a distinction between fertile and infertile males. Sperm motility and mean velocity showed the next highest diagnostic rate. On the other hand, sperm concentration showed a poor diagnostic rate (36.8%). In addition, there was no significant correlation between penetration rate and any other seminal parameters. These results suggest that the SPA will be an essential test for evaluating male fertility and penetration rate may be a marker of male fertility in the treatment of male infertility.

Adult

The use of in vitro fertilization to detect reductions in the fertility of male rats exposed to 1,3-dinitrobenzene.

1,3-Dinitrobenzene (DNB) is an intermediate chemical in the manufacture of dyes and explosives and its toxic effects include specific damage to the Sertoli cells of the testis. This investigation determined the effect a toxic insult to Sertoli cells had on the functional capacity of developing germ cells as assessed by in vitro fertilization. Male rats were given a single, oral dose of 5, 15, or 25 mg DNB/kg. At selected times after treatment, spermatozoa recovered from the cauda epididymidis were tested for fertilizing capacity using in vitro fertilization techniques and the testicular response to DNB was determined by histological examination. Treatment with 15 and 25 mg DNB/kg resulted in substantial exfoliation of germ cells between 0.5 and 3.5 weeks after exposure and again after 4.5 weeks; seminiferous tubules which were not depleted showed signs of disrupted spermatogenesis. Reduced sperm fertilizing capacity in vitro was observed from 1.5 to 5 weeks and between 7.5 and 8.5 weeks after treatment with 15 and 25 mg DNB/kg. There were slight, but significant, reductions in fertility at 3, 5.5, 7.5, and 8.5 weeks after dosing with 5 mg DNB/kg. These data suggested that DNB did not affect all Sertoli cells equally, but acted in a stage-specific manner. Stages III, IV, XII, and XIV were most vulnerable to the toxicant. Germ cells associated with an affected Sertoli cell were usually sloughed off, resulting in lowered fertility at the time when these cells should have reached maturity in the epididymis. The extent of the testicular lesions and the loss of fertility were dose dependent. This investigation confirmed the use of in vitro fertilization to detect the effects of testicular toxicants.

Animals

Relationship of antisperm antibodies to oocyte fertilization in in vitro fertilization-embryo transfer.

Antisperm antibodies (ASA) appear to impair reproduction; however, their clinical significance in in vitro fertilization-embryo transfer (IVF-ET) is unestablished. For examination of this question, the immunobead binding technique was used to identify IgA, IgG, and IgM ASA in the serum, semen, and follicular fluid (FF) of 40 couples undergoing IVF-ET. ASA binding to sperm tail tip did not predict the fertilization rate of uniformly inseminated mature oocytes. Similarly, ASA binding to sperm head in semen and male serum did not predict fertilization. However, the fertilization rate in couples with ASA to sperm head (ASA-H) of at least one isotype in female serum (n = 6) was significantly less than in those without ASA-H (n = 34; 34% versus 74%, P less than 0.01). Among these women, oocyte fertilization rates were 33% versus 71% (P less than 0.001). Sixty percent of women whose ova did not fertilize (n = 5) had ASA-H in their serum versus 6% of those whose ova did (n = 35; P less than 0.05). The presence of ASA-H in FF also correlated with fertilization. ASA-H in female serum reduced the zygote cleavage rate from 91% to 67% (P = 0.51). We conclude that the presence of ASA-H in female serum and FF is associated with reduced fertilization in IVF-ET.

Antibodies

Modification of the zona-free hamster ova bioassay of boar sperm fertility and correlation with in vivo fertility.

These studies were designed to evaluate the ability of the zona-free hamster ova bioassay to detect differences in fertility of boar sperm. In the first study, sperm from two previously infertile boars were compared to sperm from seven previously fertile boars. The percentage of zona-free hamster ova penetrated by sperm from the previously infertile boars was significantly lower than the percentage of ova penetrated by sperm from previously fertile boars (18% of ova penetrated vs. 83%, P less than .001). In the 14 ejaculates from the previously infertile boars that had ejaculate motilities of 50% or greater, the percentage of zona-free hamster ova penetrated continued to be lower than in ejaculates from the fertile boars. One of the two previously infertile boars consistently had a normal semen analysis. The only two observed manifestations of his reduced fertility were his zero conception rate and the limited ability of his sperm to penetrate zona-free hamster ova. In the second study, females were inseminated with equal numbers of sperm from two previously fertile males and the paternity of offspring determined at birth. The experiment was replicated with four combinations of six boars. A high correlation was observed between the percentage of offspring sired and the ability to penetrate zona-free hamster ova (R = .89). Neither morphology nor the ability of the sperm to undergo an acrosome reaction during in vitro incubation was correlated with fertility in the competitive mating situation. These results suggest the zona-free hamster ova bioassay can improve the in vitro fertility assessment of fresh boar semen.

Animals

Heifer fertility and its relationship with cow fertility and production traits in Holstein dairy cattle.

Breeding receipts from three AI units were merged with Ontario Dairy Herd Improvement Corporation and Record of Performance production records. Data comprised 53,705 heifer, 41,253 lactation 1, 14,688 lactation 2, and 3054 lactation 3 records by daughters of 2150 sires represented in 15,877 herd-year-seasons of birth. Three measures of heifer fertility, three measures of cow fertility, and three measures of production were investigated. Measures of heifer fertility were ages at first and last breeding and number of inseminations per conception. Cow fertility traits were days from calving to first breeding, days open, and number of inseminations per conception. Production traits were breed class average milk, breed class average fat, and fat percentage. Relationships among these nine traits for the first three lactations were estimated using a maximum likelihood multiple-trait procedure. The linear mixed model for each trait included fixed effects of herd-year-season of birth and genetic groups of sire and the random effect of sire. Transformations of the data for nonnormality had no influence on the estimates of genetic and phenotypic parameters. The heritability of .12 for age at first insemination, which was higher than other heifer fertility traits, indicated that selection would result in genetic response. Genetic and phenotypic correlations between heifer fertility and cow fertility and production traits in all three lactations were not different from zero. There was no genetic antagonism between fertility and subsequent production traits.

Animals

[Comparative study of the morpho-functional features of the sperm of fertile and infertile men and their relation to fertilizing ability].

The recent advances in assisted reproduction procedures have helped to the better understanding of the female reproductive physiology and pathology, however, male infertility remains as a poor explained medical problem, nevertheless it occurs in almost 50% of infertile couples. Oligoasthenozoospermia is one of the more common causes of male infertility, therefore we measured in 10 patients with this diagnosis and in 10 fertile euspermic men, besides the parameters included in the standard semen analysis, the quantitative motility (photography method), capacitation-acrosome reaction and the sperm ability to fertilize zona-free hamster oocytes, with the aim to correlate the morphofunctional characteristics of the male gametes with their fertilizing capacity. The results showed significant differences in every parameter studied, including the correlation analysis. In relation with the in vitro induction of the acrosome reaction in both groups, we found significant correlations of the sperm fertilizing ability and the progressive sperm motility with this parameter (fertile group: RS = 0.834, P less than 0.005 & RS = 0.612, P less than 0.05; infertile group: RS = 0.986, P less than 0.001 & RS = 0.536, P less than 0.05 respectively), nevertheless the sperm rate which completed this process was low in relation to the total sperm population even in the fertile men (9.4 +/- 2.0% & 4.4 +/- 2.5% acrosome reacted cells after 18 h of incubation, in the fertile and infertile males respectively). The results also showed the presence of fully capacitated spermatozoa in both groups, since they penetrated the zona-free hamster eggs and decondensed their chromatin (73.9 +/- 13.4% & 10.4 +/- 7.7% penetrated eggs in the euspermic and oligoasthenozoospermic individuals respectively), however, the spermatozoa from the oligoasthenozoospermic men showed low polyspermy indexes too (0.1 penetrated spermatozoa/inseminated oocyte). In this last group we found, in addition, that the mean sperm velocity and the abnormal sperm morphology rate showed significant correlations with the fertilizing ability of the male gametes too (RS = 0.986, P less than 0.005 & RS = -0.942, P less than 0.005. respectively). These data allow us to suggest that before an infertile man is involved in any assisted reproduction program, the presence of possible morphofunctional alterations in the spermatozoa be analyzed, with the aim to be able to make a better prognosis about the success with these patients.

Acrosome

Evaluation of bull semen fertility by homologous in vitro fertilization tests.

In vitro fertilization assays were performed to investigate their validity in evaluating artificial insemination (AI) bull fertility. A total of 1,532 oocytes, collected from ovaries at the abattoir, were subsequently used in a 4 x 6 x 2 factorial design: 4 doses of heparin added into the capacitation and fertilization medium (0; 0.05; 0.1 and 0.2 micrograms/ml), 6 different bulls with known on-field non-return (NR) rates (range: 64.6-75.3%) and 2 different ejaculates for each bull, collected within a approximately 1-month interval. Oocytes were considered fertilized when 2 pronuclei (or more) were seen in the ooplasm. Both the heparin dose and bull exerted a highly significant effect on the in vitro fertilization (IVF) rates which ranged, per oocyte group, from 30-80%; bull x dose of heparin interaction was significant (P less than 0.001). The 0.05 micrograms/ml dose of heparin was optimal for discriminating individual bulls. At that dose, the correlation coefficients between the bulls, NR rates and the IVF rates from each ejaculate (within-bull or the mean of two ejaculates), were highly significant (r = 0.83). The rates of polyspermy were also significantly influenced by bull and heparin dose, but there was no interaction. In conclusion, capacitation and fertilization in a modified Tyrode medium containing 0.05 micrograms/ml of heparin may be a valuable tool for evaluating AI bull fertility.

Animals

Failure of fertilization in in vitro fertilization: the "occult" male factor.

Failure of fertilization in patients undergoing in vitro fertilization (IVF) deserves extensive analysis for better prediction of the success or failure of this therapeutic modality. Consequently, we retrospectively studied the 52 couples in whom fertilization failed during Norfolk series 18 to 25, in an effort to establish the precise causes of failure. In the initial evaluation, pure oocyte abnormalities were identified in 19.2% of the cases; 32.6% showed sperm abnormalities, and a combination of oocyte and sperm anomalies was found in 7.7%. In 40.4% of the cases, failure of fertilization could not be explained. Reassessment of sperm morphology by new, strict criteria increased the identification of sperm abnormalities to 61.5% and of combined sperm and oocyte anomalies to 13.4%, for a total of 74.9% of sperm factors involved, as opposed to 40.3% in the original evaluation. The incidence of unexplained failed fertilization was substantially reduced, to 11.5%. In a control group (tubal infertility) matched by age, date, and stimulation, in whom fertilization occurred, 83.3% had normal sperm parameters as judged by the new criteria for morphology evaluation. This paper emphasizes the need for a more accurate diagnosis of sperm abnormalities to establish the true incidence of this factor in failed fertilization and to obtain information of prognostic value to patients and clinicians.

Adult

Failed fertilization in human in vitro fertilization analyzed with the deoxyribonucleic acid-specific fluorochrome Hoechst 33342.

The degree and normality of nuclear maturation were assessed with the fluorochrome Hoechst 33342 in two groups of inseminated human oocytes that had failed to undergo fertilization. Group 1 consisted of 67 oocytes from 27 patients, each of whom had at least two other oocytes that had been fertilized and had cleaved. Group 2 consisted of 65 oocytes from 14 patients, none of whose oocytes had been fertilized. In group 1, 52.3% of the oocytes were found to be immature (germinal vesicle stage or metaphase-telophase I), whereas in group 2 only 26% were found to be immature. Thus oocyte nuclear immaturity was the major cause of fertilization failure when companion oocytes were fertilized. When no oocytes of a patient were fertilized, most oocytes were found to be mature, so other factors, such as sperm dysfunction or zona binding abnormalities, must account for most of the fertilization failure in this group of patients.

Benzimidazoles

Delayed fertilization during in vitro fertilization and embryo transfer cycles: analysis of causes and impact on overall results.

This study evaluated possible causes of delayed fertilization during in vitro fertilization (IVF) cycles, its repetitiveness, and its influence on IVF results in 23 patients (27 cycles) with delayed fertilization of greater than or equal to 1 preovulatory oocyte(s). In 15 cycles, reinsemination with husband's semen was performed at 18 hours. Possible causes of delayed fertilization were oocyte defects (10 cycles, 37.0%), sperm defects (4 cycles, 14.8%), oocyte and sperm defects (4 cycles, 14.8%), and no detectable gamete defects (9 cycles, 33.3%). Overall fertilization rate was 47.9%. No pregnancies were observed in 10 patients with one embryo transferred. Recurrence rate of delayed fertilization per patient was 17.3%; overall ongoing pregnancy rate/cycle was 10.3%. Although repetitiveness of delayed fertilization is low, it seems to impact negatively on IVF results.

Embryo Transfer