Patients Undergoing IVF Treatment Research Articles

Cryopreservation of human failed maturation oocytes shows that vitrification gives superior outcomes to slow cooling

This study investigated whether failed maturation oocytes could be used to evaluate different cryopreservation procedures. A total of 289 failed maturation oocytes (GV and MI stages), obtained from 169 patients undergoing IVF treatment (mean age 33.84 ± 5.0) were divided into two different slow-cooling groups (1.5 mol/l 1,2-propanediol + 0.2 mol/l sucrose in either NaCl (group A) or choline chloride (ChCl) (group B) based cryopreservation solutions) and one vitrification group (15% ethylene glycol + 15% dimethyl sulphoxide). Survival rate, in vitro maturation (IVM) rate, fertilization and developmental rate of cryopreserved oocytes were assessed. Regardless of the stage at which cryopreservation was performed (GV + MI), the slow cooling with ChCl based medium always gave significantly lower survival rate than the slow cooling in NaCl based medium ( p = 0.01) and vitrification ( p < 0.001). An extended study also showed statistically reduced survival rate between slow-cooling NaCl based medium and vitrification ( p < 0.05). Global results of in vitro maturation and fertilization showed worse results between both slow-cooling NaCl and ChCl based media versus vitrification. In conclusion, for oocytes that had failed to mature, vitrification gave better survival, maturation, fertilization and also cleavage rates than the slow-cooling protocols. Four cells embryos were obtained only from vitrified in vitro matured MI oocytes.

Ganirelix for luteolysis in poor responder patients undergoing IVF treatment: a Scandinavian multicenter ‘extended pilot study’

To enhance oocyte yield and pregnancy outcome in poor responder women undergoing IVF treatment, daily low dose GnRH antagonist administration was given during the late luteal phase to induce luteolysis and possibly secure a more synchronous cohort of recruitable follicles. An open extended pilot study in four Scandinavian fertility centers was done including 60 patients. Poor response was defined as when < or = 5 follicles developed in a preceding cycle following a long agonist protocol with the use of > 2000 IU FSH. GnRH antagonist (ganirelix) was given, 0.25 mg s.c. daily, from days 3 to 5 before expected start of menstruation and continued for 4-7 days. On cycle day 2-3 a starting dose of rFSH (300-400 IU/day) was given. At a leading follicle diameter of 14 mm, ganirelix administration was resumed until final oocyte maturation was induced with 10,000 IU hCG. GnRH antagonist only marginally affected the intercycle FSH rise; basal levels of FSH remained similar to those seen after 4 days of antagonist administration. The protocol effectively induced low LH levels and luteolysis, but daily administration of 350 IU rFSH (median) for 11 days only led to the collection of 3 oocytes in 49 oocyte retrievals resulting in 5 pregnancies (4 delivered). Despite GnRH antagonist administration in the late luteal phase and menstrual bleeding, FSH was not sufficiently reduced to secure a more synchronic cohort of recruitable follicles. Novel GnRH antagonists more specifically targeting FSH release may improve the stimulation results in poor responders.

Live births after management of severe OHSS by GnRH antagonist administration in the luteal phase

Ovarian hyperstimulation syndrome (OHSS) is a serious complication of ovarian stimulation protocols. Currently, no curative therapy exists and the main preventive option is cycle cancellation. Gonadotrophin-releasing hormone (GnRH) antagonist administration in the luteal phase was recently proposed as a new approach for the management of patients with established severe OHSS. Three polycystic ovarian syndrome patients undergoing IVF treatment developed severe OHSS, diagnosed 6 days after oocyte retrieval. On day 6, the patients underwent blastocyst transfer and received GnRH antagonist for 4 days, combined with luteal phase support using exogenous oestradiol and progesterone. Two patients had successful pregnancies that resulted in births of healthy infants, while one patient had a biochemical pregnancy. In all patients, established severe OHSS regressed to a moderate form of the syndrome, no pregnancy-induced life-threatening OHSS was observed, while a short monitoring period was required at an outpatient level, avoiding the need for patient hospitalization. This is the first report in the literature on GnRH antagonist administration in the luteal phase, combined with embryo transfer and exogenous oestradiol and progesterone supplementation. This novel treatment was effective in the regression of established severe OHSS, and resulted in the birth of healthy infants.

Appropriate timing of uterine cavity length measurement positively affects assisted reproduction cycle outcome

An appropriate and easy embryo transfer has a direct impact on pregnancy rates. Proper evaluation of the uterocervical axis and uterine depth are necessary for suitable embryo transfer. The aim of this study was to assess the appropriate time for cervical axis evaluation and uterine measurement. A total of 124 patients undergoing IVF treatment were included in the study. They were divided equally into two groups. In group I (62 women), uterine cavity depth was measured and the uterocervical axis was determined on day 2 or 3 of the menstrual cycle, and in group II (62 women) at the time of oocyte retrieval. There was a statistically significant difference in clinical pregnancy rates between the two groups (P = 0.006). Thirty-four women became pregnant in group I (64.2%) versus 19 women in group II (35.8%). In conclusion, uterine cavity measurement is necessary for suitable embryo transfer. It seems that the time of measurement significantly affects clinical pregnancy rate in IVF cycles. The best time for uterine measurement is on day 2 or 3 of menstruation.

Comparison of follicular fluid and serum levels of Inhibin A and Inhibin B with calculated indices used as predictive markers of ovarian hyperstimulation syndrome in IVF patients

BackgroundOvarian Hyperstimulation Syndrome (OHSS) is a severe health complication observed in some patients undergoing hormonal stimulation during IVF. Presence of OHSS is often associated with a high count of growing follicles responding to FSH hyperstimulation. However, the number of responding follicles may not be sufficient enough to predict the onset and severity of OHSS. The aim of this study was to find whether follicular fluid (FF) and serum concentrations of Inhibin A and Inhibin B in patients undergoing IVF treatment may serve as a predictor of OHSS status independent of the growing follicles count.MethodsSerum and follicular fluid of fifty-three women undertaking the IVF program were separated into four groups according to their OHSS status and growing follicles count and analyzed for serum and FF concentrations of Inhibin A and Inhibin B. The resulting data were combined with clinical and demographic data to calculate indices independent of the growing follicles count.ResultsSerum Inhibin A and Inhibin B concentrations showed no significant difference between the severe OHSS group and the control group without OHSS. Moreover, the serum concentrations of Inhibin A and Inhibin B were strongly correlated with the growing follicles count. Their concentrations in the high responders group (>18 follicles) were significantly higher (p < 0.00001, p < 0.0001) when compared with normal and low responders (<18 follicles). To suppress the dependence on the growing follicle count, three indices were constructed and calculated. The best association with OHSS status and independence of the growing follicle count was achieved by using the Inhibin B TFF/SBM index calculated as follows: [concentration in FF] × [growing follicle count]/[concentration in serum] × [body mass]. The Inhibin B TFF/SBM index showed a clear difference (p = 0,00433) between the group with severe OHSS and the control group, while showing no apparent correlation with the growing follicle count.ConclusionThese observations demonstrated that while neither serum nor FF concentrations of Inhibin A nor Inhibin B can be used as an OHSS predictor independent of the growing follicle count, calculated indices may meet the criteria.

Usage of herbal medications in patients undergoing IVF treatment in an Irish infertility treatment unit

Usage of herbal and complimentary medicines by patients presenting for procedures that require the use of anaesthetic agents is common. To determine the prevalence of herbal medication usage among female patients attending for assisted reproduction procedures involving the use of sedative or hypnotic agents. Questionnaire-based survey carried out over a 6-month period from January to July 2007. Forty-six percent of patients admitted regular use of herbal medications, with 38% of patients having taken herbal preparations in the 3-month period prior to their attendance for treatment. No patient taking herbal preparations had discussed the use of these products with the attending anaesthetist or fertility specialist prior to attending for the procedure. The usage of herbal medications with potential for serious adverse effects is high in this unique patient population. Education of both healthcare providers and patients is indicated.

Visfatin and leptin levels in women with polycystic ovaries undergoing ovarian stimulation

To detect the levels of visfatin and leptin in the serum as well as in the follicular fluid (FF) of polycystic ovary syndrome (PCOS) patients undergoing controlled ovarian stimulation and to compare them with the levels found in age- and weight-matched normally ovulating women under IVF treatment. Prospective study. Assisted Reproduction Unit, Second Department of Obstetrics and Gynecology, University of Athens, Aretaieion Hospital, Athens, Greece. Forty patients with diagnosed PCOS and 40 age- and weight-matched non-PCOS control women enrolled in the IVF program. Blood and FF samples were collected from all subjects at oocyte retrieval. Visfatin and leptin levels were measured in serum and FF using ELISA. Serum visfatin levels were significantly increased in women with PCOS, whereas FF visfatin levels, which were lower than serum levels, did not differ between groups. Serum leptin levels did not differ between groups and were lower than FF levels. Women with polycystic ovaries exhibit significantly increased serum visfatin and decreased FF leptin levels compared with control subjects of similar age and body mass index, indicating a probable role for visfatin in the general state of insulin resistance and a local contribution in the follicle for leptin in patients undergoing IVF treatment.

Association between sperm cell chromatin condensation, morphology based on strict criteria, and fertilization, cleavage and pregnancy rates in an IVF program.

In this study, a total of 95 ejaculates from infertile patients were investigated morphologically according to Kruger's strict criteria and 78 of the 95 ejaculates were stained for chromatin condensation with acidic aniline blue. Patients were divided into two groups based on the percentage of morphologically normal spermatozoa as follows: Men with normal sperm morphology < 14% (Group 1), and men with normal morphology > 14% (Group 2). The relationship between percentage of normal sperm morphology and fertilization, cleavage and pregnancy rate was analysed. The rates were 33.7%, 57.1% and 0.0% respectively, in the first group. The corresponding values for the second group were 76.1%, 68.2% and 32.1%. The fertilization and pregnancy rates correlate significantly with morphologically normal spermatozoa. In regard to the percentage of morphologically normal spermatozoa stained with aniline blue, patients were divided into two groups: patients with 0-20% stained spermatozoa (Group I) and those with > 20% (Group 2). Fertilization and pregnancy rates were higher in the first group than in the second group (79.9%, 52.8% vs. 58.8%, 29.5%). The results demonstrate that chromatin condensation visualized by aniline blue staining is a good predictor for IVF outcome and should be considered besides morphology by sperm assessment for patients undergoing IVF treatment.

Vitrification is a highly efficient method to cryopreserve human embryos in in vitro fertilization patients at high risk of developing ovarian hyperstimulation syndrome

This study aimed to evaluate the efficacy of embryo vitrification as an emergency procedure for patients at high risk of developing ovarian hyperstimulation syndrome (OHSS). A total of 69 embryos, derived from 24 patients for whom embryo transfer could not be performed because of the risk of developing OHSS, were vitrified and warmed for deferred embryo transfer. Surviving embryos were transferred, resulting in 10 clinical pregnancies, of which 4 were successfully delivered and the remaining 6 are still ongoing.

Comparative genomic hybridization of oocytes and first polar bodies from young donors

Chromosome abnormalities are common in oocytes derived from patients undergoing IVF treatment. The proportion of oocytes displaying aneuploidy is closely related to maternal age and may exceed 60% in patients over 40 years old. However, little information currently exists concerning the incidence of such anomalies in oocytes derived from young fertile women. A total of 121 metaphase II oocytes and their corresponding first polar bodies (PB) were analysed with the use of a comprehensive cytogenetic method, comparative genomic hybridization (CGH). The oocytes were donated from 13 young women (average age 22 years) without any known fertility problems. All oocytes were mature at the time of retrieval and were unexposed to spermatozoa. A low aneuploidy rate (3%) was detected. These results clearly indicate that meiosis I segregation errors are not frequent in oocytes of young fertile women. The higher aneuploidy rates reported in embryos derived from donor oocytes could be due to aggressive hormonal stimulation, in combination with male factors. However a definite contributing factor remains to be elucidated. The data obtained during this study also illustrate that CGH accurately and efficiently detects aneuploidy, confirming that it is suitable for application in a clinical setting for the assessment of oocytes, via PB analysis.

Meiotic spindle recovery is faster in vitrification of human oocytes compared to slow freezing

To investigate spindle behavior during and after slow freezing at room temperature (RT) and vitrification at different temperatures. Randomized, comparative study. University hospital. Patients undergoing IVF treatment volunteered for the study and donated part of their supernumerary oocytes. Metaphase II oocytes were divided into group A: slow freezing RT /thawing RT; group B: vitrification RT/warming RT; group C: vitrification RT/warming 37 degrees C; and group D: vitrification 37 degrees C/warming 37 degrees C. Spindle presence was evaluated at each step of the four procedures and in culture. Cumulative spindle recovery rate comparing warming phase of the three vitrification groups and culture phase among the four groups. During warming, the three vitrification groups showed a significantly fast spindle recovery rate compared to the thawing of the slow freezing group. A progressively significant fast cumulative recovery rate was observed in the three vitrification groups by increasing the number of phases at physiological temperature (hazard rate = 2.68; 95% confidence interval 1.71-4.02). The present study demonstrates that spindle recovery is faster in vitrification than in slow freezing. These data support a possible protective effect of vitrification/warming at 37 degrees C on the meiotic spindle structure and, therefore, on the subsequent clinical outcome of the procedure, although comparative clinical studies are needed.

Levels of steroidogenic acute regulatory protein and mitochondrial membrane potential in granulosa cells of older poor-responder women

To compare mitochondrial function in granulosa cells obtained from older (>40 y) low-responder IVF patients with that of young (<35 y) good-responder patients. Prospective laboratory research. In vitro fertilization unit in a university hospital. Twenty patients undergoing IVF treatment cycles. Ultrasound guided oocytes pick-up. Mitochondrial function examined by using JC-1 stain for the mitochondrial membrane potential in granulosa cells of both groups and Western blots for assaying and quantification of steroidogenic acute regulatory protein (StAR) and p450scc (side-chain cleavage). The number of granulosa cells per follicle differed between the two groups, with fewer granulosa cells isolated in the older low-responder women, compared with in the young, normal responders who were the control women. Trypan blue-negative cells showed similar undisturbed mitochondrial membrane potential, and similar ratios of apoptotic granulosa cells were observed in the two groups. In addition, there was no difference in StAR and P450scc protein levels between the two groups. Our results demonstrate a significant decrease in the number of total aspirated granulosa cells per follicle in older, poor-responder women, which probably explains the reduced hormonal production by those follicles. However, those cells demonstrate normal mitochondrial membrane potential as well as similar levels of StAR, P450scc, and de novo steroid hormone synthesis in the two groups of patients. Our results do not support mitochondrial dysfunction as a main mechanism of reproductive aging.

Women with poor response to ovarian stimulation have increased follicular bone morphogenetic protein-15 levels

To evaluate the levels of bone morphogenetic protein-15 (BMP-15) in human follicular fluid (FF) and its association with response to ovarian stimulation. Western blotting was performed to determine the levels of BMP-15 in FF obtained from follicle aspirates in 70 patients undergoing IVF treatment. According to the response to ovarian stimulation the patients were divided into poor responder group and normal responder group. BMP-15 levels in FF of poor responders were significantly higher than those in normal responders (1.01 +/- 0.34 vs 0.77 +/- 0.24, P<0.01). Increased levels of BMP-15 in FF may be associated with poor response to ovarian stimulation.

Parental influence on multinucleation in early human embryos

Multinucleation in human embryos, especially on day 2 of development, has been described as a strong positive predictor of implantation and is routinely monitored in IVF labs. We pretend to find out whether human embryo multinucleation depends on any parental factor or if it appears haphazardly. Retrospective study of patients under IVF treatment. We selected in our database a total of 1548 cycles of patients undergoing IVF treatment and 540 cycles from our oocyte donation program from 2004 to 2006 in which we checked the multinucleation in day 2 and day 3 in a total of 15581 embryos. We recorded for each embryo if nuclei were visible or not at all, and, when visible, the number of nuclei in each of the blastomeres. We calculated the proportion of these types of embryo per cycle (“mononucleated”, “multinucleated” or “not visible”). In total, 35% of the embryos were mononucleated on day 2, 16% were multinucleated and 49% did not have visible nuclei. The distribution of the proportion of visible embryos per cycle is not normally distributed. Most cycles (75%) have a normal distribution of ‘not visible’ embryos per cycle with a random 50% chance of been visible per embryo, but there are other two groups of cycles where all embryos were visible (10%) or not visible (15%) regardless of the number of oocytes retrieved. Embryos from the oocyte donation program showed higher proportion of mononucleated embryos (37% vs. 32% P<0.01) and lower of multinucleated embryos (13% vs. 18% P<0.01) whereas no difference in the proportion of ‘not visible’ embryos was observed (49% vs. 50% P=0.72). We compare the pregnancy rates in three groups: cycles with all embryos mononucleated, cycles with all embryos multinucleated and cycles with any other combination. In the oocyte donation program, no difference in pregnancy rate was found among the three groups (57%, 49%, 59% P=0.47). While on the contrary, in the standard program the multinucleated group showed lower pregnancy rates (58%, 41%, 62% P<0.001). As previously described, multinucleation on day 2 correlates with lower pregnancy and implantation rates. In this study we found that multinucleation has less impact on oocyte donation cycles. Most probably multinucleation by itself is not enough to discard an embryo but worsens embryo quality in addition to other factors not present in donor oocytes. In some cases, the presence of many multinucleated embryos is a patient characteristic which repeats in subsequent cycles.

The effect of luteal phase vaginal estradiol supplementation on the success of in vitro fertilization treatment: a prospective randomized study

To determine whether the use of luteal phase vaginal E(2) supplementation improves clinical pregnancy rates in patients undergoing IVF treatment. Prospective randomized controlled trial. University-based tertiary fertility center. One hundred sixty-six patients undergoing their first cycle of IVF treatment. Patients underwent three different protocols for controlled ovarian hyperstimulation for IVF treatment with long GnRH agonist suppression, use of GnRH antagonist, or a microdose GnRH agonist protocol. Luteal phase support was in the form of IM P. Patients randomized into the study group (n = 84) received E(2) supplementation in the form of vaginal estrace 2 mg twice a day starting on the day of ET. Patients randomized to the control group (n = 82) received no E(2) supplementation. Clinical pregnancy rates. There were no significant differences in the implantation (56/210 [26.7%] vs. 64/203 [31.5%]), clinical pregnancy (42/84 [50%] vs. 52/82 [63.4%]), and ongoing pregnancy rates (40/84 [47.6%] vs. 46/82 [56.1%]) between the study and control groups, respectively. In the subgroup of patients who used the long GnRH agonist suppression protocol, there was a lower clinical pregnancy rate in the study group compared with the control group (27/55 [49.1%] vs. 42/59 [71.2%]). There were, however, no differences in clinical pregnancy rates between the two groups in patients who used either the GnRH antagonist or microdose GnRH agonist protocols. The addition of vaginal E(2) supplementation to routine P supplementation for luteal support does not improve the probability of conception after IVF treatment.

Getting pregnant after tubal sterilization: surgical reversal or IVF?

When women regret having had a tubal sterilization, is the pregnancy rate higher with surgical reversal or IVF? This retrospective cohort study analyses the delivery rates of 163 patients undergoing IVF treatment (n = 79) or surgical reversal (n = 84). Pregnancy outcomes were obtained by reviewing medical records or contacting private physicians and patients. The life table method was used to calculate the chance of becoming pregnant and to construct cumulative pregnancy curves. Cumulative pregnancy curves are compared by log rank tests. A P-value of <0.05 is considered as statistically significant. The cost-effectiveness of the two strategies was also evaluated. Patient characteristics did not differ between the two groups. The cumulative delivery rate during 72 months was 52.0% in the IVF group and 59.5% in the reversal group (ns). Age was the only factor that influenced delivery rates significantly. The cumulative delivery rate for patients aged < 37 years was 52.4% after IVF and 72.2% after reversal (P = 0.012), while cumulative delivery rates for patients aged 37 years or older were, respectively, 51.4 and 36.6%, a difference that did not reach statistical significance. The average cost per delivery was 11,707 euros for IVF, compared with 6015 euros for surgical reversal. However, in patients aged 37 years or older the difference in cost was smaller. Considering the cumulative delivery rates involved, surgical reversal is recommended for patients younger than 37; older patients are advised to opt for IVF.

P-798: Induction of oocyte maturation with GnRH agonist in high-risk patients undergoing IVF treatment does not adversely affect implantation rate

Previous studies have suggested that GnRH agonist trigger during IVF treatment may adversely affect implantation rates. This may be due to inadequate luteal phase and early pregnancy steroidal concentrations induced by early luteolysis. We evaluated the effect of GnRH agonist trigger on implantation and pregnancy rates in high-risk patients who used a different luteal phase steroidal supplementation protocol than that used in previous studies.

Mitochondrial DNA Deletion in Human Oocytes

To compare the incidence of mitochondrial DNA (mtDNA) deletion in normal and unfertilized oocytes. Laboratory study. Metaphase II oocytes were obtained during IVF cycles. Normal oocytes were obtained from women undergoing IVF treatments because of non-obstructive azoospemia but no sperm could be retrieved from testicular biopsy. The other group of oocytes came from patients undergoing IVF treatment but the oocytes were unfertilized. The presence of mtDNA in single human oocyte was confirmed by standard PCR with a set of primers (forward primer L467 and reverse primer H725) for amplifying a fragment outside the hotspot of deletion area. The nested PCR reaction was employed to detect the presence of mtDNA deletions, and was carried out on those oocytes with a positive band from the first PCR reaction. The two pairs of primers (outer primers L8282 + H13650 and inner primers L8334 + H13578) flanked the region of the K5S mtDNA deletion. If the K5S deletion was present, the nested PCR product was 256 bp long. In the absence of the deletion, there was no DNA amplication. All experiments included water-only negative controls and a positive control. The PCR products were separated on 2% agarose gel stained with ethidium bromide. PCR products were confirmed by DNA sequencing. Thirty normal and 155 unfertilized MII oocytes were obtained from 4 and 62 donors respectively. The incidence of K5S mtDNA deletion in the normal oocytes was 23.3% which was statistically not different from the 34.6% in the unfertilized oocytes (p = 0.228). When the comparison was confined to donors of unfertilized oocytes < 35 years of age (n = 97), the incidence of K5S mtDNA deletion remained comparable (p = 0.626) between the two groups. When only the unfertilized oocytes were studied, women ≥= 35 years of age had a significantly higher incidence of K5S mtDNA deletion (46.6%) when compared with younger women (27.8%) (p = 0.018). The incidence of mtDNA deletion in unfertilized oocytes was similar to that of the normal oocytes, but was higher in women with advanced age. These findings were in concordance with the observation that the rate of accumulation of somatic mtDNA mutations increased during the aging process and hence the incidence of mtDNA deletion can reflect ovarian aging.

Rapid Effects of Pesticides on Intracellular Calcium Changes in Human Granulosa-Lutein Cells

Rapid Effects of Pesticides on Intracellular Calcium Changes in Human Granulosa-Lutein Cells

Prognostic value of some ovarian reserve tests in poor responders

The objective of this study was to determine the prognostic value of the basal estradiol (E2) and inhibin-B levels, the antral follicle count (AFC), and the clomiphene citrate challenge test (CCCT) of ovarian response in controlled ovarian hyperstimulation (COH), in an outcome with normal follicle-stimulating hormone (FSH) concentration in the early follicular phase of the menstrual cycle. Fifty-two patients undergoing IVF treatment were included in the study. Blood samples were collected for assessment of basal E2, FSH, and inhibin-B levels. Transvaginal ultrasound of an unstimulated cycle was performed to determine the mean antral follicle count (AFC). Serum FSH concentration was measured again on day 10 for CCCT performance. The mean values of women's age, and basal and day 10 FSH levels were significantly higher in cancelled cycles than in the control group, whereas basal inhibin-B and AFC were significantly higher in the latter. The mean basal E2 concentration was similar in both groups. The results from the logistic regression analysis show that CCCT (cut-off point FSH > 12.5 mIU/ml; AUCROC = 0.90) was a better single predictor of poor ovarian response than AFC (AUCROC = 0.85) and inhibin-B (AUCROC = 0.79) with a correct prediction for CCCT (86.5%), antral follicle count (84.6%), and for inhibin-B (82.7%). In women with normal basal FSH level, the determination of E2 has no prognostic value for the outcome of poor responders. However, CCCT, AFC, and inhibin-B tests, when applied separately, produce good prognostic values. CCCT is the best single predictor of poor ovarian response, followed by antral follicle count and basal inhibin-B values. In spite of that, CCCT does not add significantly to the simpler AFC ultrasound test in the prediction of poor ovarian response.