S. Al-Hasani

Three years of routine vitrification of human zygotes: is it still fair to advocate slow-rate freezing?

Cryopreservation of human oocytes and embryos is a necessary tool in assisted reproduction treatment that leads to an increased cumulative outcome while decreasing costs. Vitrification is a cryopreservation technique that leads to a glass-like solidification, with rapid cooling of cells or tissues. Nowadays vitrification is claimed to be the future of cryopreservation of human embryos due to improved survival rates and clinical outcomes. This study was conducted at a university clinic to assess the safety and efficiency of vitrification of human zygotes as a routine procedure. A total of 849 pronuclear-stage (PN) zygotes were vitrified between March 2004 and July 2006. During this period, 103 cycles of cryopreserved embryo transfer were completed. In total, 339 PN zygotes were thawed resulting in an 89% survival rate (302 PN zygotes). The mean number of embryos per transfer was 2.2. The pregnancy rate obtained was three times higher (36.9%) than that obtained with the slow-rate freezing method (10.2%) used previously in the same centre. In conclusion, vitrification of human zygotes at the pronuclear stage seems to be a successful and reliable method with favourable outcomes and can be recommended as a routine technique for cryopreservation of human embryos.

Transvaginal sonography of the endometrium during ovum pickup in stimulated cycles for in vitro fertilization.

The endometrium of a series of 190 in vitro fertilization patients was investigated by transvaginal ultrasound. The endometrial pattern was related to the likelihood of implantation. No such relationship was found for the thickness of the endometrium. There was not a strong correlation between the endometrium and hormonal values. However, three endometrial patterns were detected after hormonal stimulation. The predominant pattern consisted of an outer hyperechogenic layer and an inner hypoechogenic layer. This pattern correlated in a positive fashion with subsequent implantation. It is concluded that the texture of the endometrium at the time of ovum pickup has a prognostic value for the likelihood of implantation to occur.

Hormone profiles under ovarian stimulation with human menopausal gonadotropin (hMG) and concomitant administration of the gonadotropin releasing hormone (GnRH)-antagonist Cetrorelix at different dosages

AbstractPurpose: The premature LH surge in ART programs seems to be avoided by daily administration of the GnRH-antagonist Cetrorelix during the midcycle phase in controlled ovarian hyperstimulation with hMG. The dosage necessary for sufficient suppression of the pituitary gland is not yet defined. Methods: To elucidate this question three daily dosages (3, 1, 0.5 mg) were administered and the hormone profiles obtained as well as the number of oocytes retrieved, the fertilization rate, and the consumption of HMG were compared. Results: No premature LH surge could be observed at any of the three dosages administered. Both gonadotropins were deeply suppressed. The fertilization rates of the oocytes obtained were 45.3% in the 3-mg group, 53.1% in the 1-mg group, and 67.7% in the 0.5-mg group. The average uses of hMG ampoules were 30 in the 3-mg group, 27 in the 1-mg group, and 26 in the 0.5-mg group. Conclusions: Cetrolix, 0.5 mg/day, administered during the midcycle phase of controlled ovarian hyperstimulation with hMG is enough to prevent completely the premature LH surge. Perhaps even lower dosages would be sufficient. Regarding fertilization rates and use of hMG, the lower dosage seems to be the most favorable.

Use of frozen-thawed testicular sperm for intracytoplasmic sperm injection

OBJECTIVE To determine the feasibility of using frozen-thawed testicular spermatozoa for intracytoplasmic sperm injection. DESIGN Prospective clinical study. SETTING A university hospital. PATIENT(S) One hundred seventy-five azoospermic men participating in a routine intracytoplasmic sperm injection program. INTERVENTION(S) The men underwent testicular biopsy for cryopreservation of tissue to be used in consecutive intracytoplasmic sperm injection treatment cycles. Their female partners underwent controlled ovarian hyperstimulation for conventional IVF treatment. MAIN OUTCOME MEASURE(S) Fertilization and pregnancy rates. RESULT(S) In 77% of the patients, spermatozoa could be harvested from the testis by an open testicular biopsy technique and used for intracytoplasmic sperm injection after freezing and thawing of testicular tissue. Histopathologic evaluation revealed a Sertoli cell-only pattern in 21%, maturation arrest in 60%, and hypospermatogenesis in 19% of the patients. In 2. 9% of the patients, carcinoma in situ or a germ cell tumor was detected. In all patients, viable spermatozoa could be visualized after the tissue samples were thawed. One hundred thirty-five intracytoplasmic sperm injection treatment cycles were performed, with a fertilization rate of 45% and a clinical pregnancy rate of 30% per oocyte retrieved. CONCLUSION(S) The use of frozen-thawed testicular tissue allows ovarian stimulation of the female partner to be timed and avoids cancellation of ovum pick-up when spermatozoa cannot be retrieved.

Current aspects of blastocyst cryopreservation

Cryopreservation of human gametes and embryos has become an essential part of assisted reproduction. Successful cryopreservation of human blastocysts is increasingly relevant as extended in-vitro culture of human embryos becomes more common, permitting routine use of blastocyst transfer in IVF programmes. This reduces the number of embryos transferred, thereby reducing multiple pregnancies and maximizing cumulative pregnancy rates per oocyte retrieval. The superiority of blastocyst freezing over earlier stage freezing in terms of implantation per thawed embryo transferred improves overall expectations for the cryopreservation programme. Therefore, a reliable procedure for the cryopreservation of blastocysts is needed because, after transfer, only a small number of supernumerary blastocysts are likely to be available for cryopreservation. Since the early 1980s, two common techniques have been used in cryopreservation: the conventional slow cooling method and the more recent rapid procedure known as vitrification. Vitrification has become an attractive alternative to slow freezing, since it appears to result in significantly higher survival and pregnancy rates. The aim of this review is to focus on the cryopreservation of human blastocysts using slow and rapid protocols and to assess the impact of the crypreservation protocol used on the survival, implantation and pregnancy rates.

Comparison of reactive oxygen species concentration in seminal plasma and semen parameters in partners of pregnant and non-pregnant patients after IVF/ICSI

The aims of this study were (i) to determine and compare the concentration of reactive oxygen species (ROS) and total antioxidant status (TAS) in seminal plasma and sperm parameters of the male partners of patients undergoing IVF or intracytoplasmic sperm injection (ICSI) treatment and (ii) to establish the relationship between ROS and TAS concentrations and sperm quality and their effect on fertilization and pregnancy rate of patients who achieved a pregnancy and those who were unsuccessful. Twenty-six IVF and 22 ICSI patients were included in this study. The ROS concentration in seminal plasma and sperm concentration, vitality (eosin test), motility, morphology, membrane integrity (HOS test), maturity (chromomycin, CMA3) and DNA fragmentation (TUNEL) results and their relationship to fertilization and pregnancy were analysed. ROS concentrations were similar regarding the seminal plasma of male partners of patients who achieved a pregnancy and those who were unsuccessful. The other semen parameters, concentration, motility, vitality, membrane and DNA integrity, were comparable in both groups. However, both groups demonstrated a negative correlation between ROS concentration and sperm vitality, membrane integrity and morphology. Moreover, an inverse correlation was found between TUNEL, vitality, and membrane integrity. In conclusion, ROS concentration in seminal plasma affects the quality of spermatozoa. A negative correlation between the ROS concentration in seminal plasma and fertilization rate in both IVF/ICSI programmes was shown.

Polyspermy in in Vitro Fertilization of Human Oocytes: Frequency and Possible Causes

In an IVF program a total of 585 oocytes (180 patients) were examined for the presence of pronuclei 16 to 20 hours after the addition of spermatozoa. The overall fertilization rate was 71%, and in 58 (10%) of the fertilized oocytes, three or more pronuclei, indicating a failure of the block to polyspermy, could be observed. The frequency of polyspermy was related to the maturity of the oocyte, determined according to morphologic criteria. Immature oocytes showed a higher percentage of polyspermic fertilization (32%) compared to that of mature oocytes (6%). Preincubation of oocytes (for 0.5-1.5, 2-4, and 5-8 hours) prior to the addition of spermatozoa increased the fertilization rate (to 67%, 70%, and 83%, respectively). The polyspermy rate, however, was not significantly different between the various preincubation intervals (13%, 14%, and 19%, respectively). The polyspermy rate was affected by the number of spermatozoa used for in vitro fertilization. Insemination with 0.5-0.8, 1.0, or 1.5-2.0 X 10(6) spermatozoa/oocyte resulted in a polyspermy rate of 6%, 20%, and 32%, respectively. The appearance of polyspermic fertilization was not related to the age of the patient (which ranged from 20 to 45 years) nor to the method of ovarian stimulation (clomiphene, hMG, or clomiphene/hMG). Because of the high incidence of polyspermy under in vitro conditions it seems to be important to routinely examine the oocytes in the pronuclear stage. Reduction of the number of spermatozoa used for in vitro fertilization and the exact timing of insemination according to the maturity of the oocyte might reduce the occurrence of polyspermic fertilization.

DNA damage of human spermatozoa in assisted reproduction: origins, diagnosis, impacts and safety

Sperm DNA contributes half the offsprings genomic material and abnormal DNA can lead to derangements in the reproductive process. Normal sperm genetic material is required for successful fertilization, as well as for further embryo and fetal development that will result in a healthy child. Thus, the damage to sperm DNA is critical in assisted reproductive techniques which are increasingly used to treat infertile couples. There has been improving data about the effects of human sperm DNA damage or fragmentation. As well, increasing knowledge concerning the effects of DNA damage on embryo and fetal development has been attained. This review aims to summarize the present knowledge on the impact of human sperm cell DNA damage on male infertility and outcome in the context of safety.

Luteal phase support using either Crinone® 8% or Utrogest®: results of a prospective, randomized study

The Crinone 8% preparation makes it possible to administer natural progesterone (90 mg) vaginally once daily for luteal phase support (LPS). Until now, no prospective, randomized studies have directly compared this new preparation with widely used Utrogest capsules, which were originally designed for oral administration but are used routinely as a vaginal preparation. A prospective, randomized study investigated 126 patients undergoing cycles of in vitro fertilization (IVF) and IVF/intracytoplasmic sperm injection (ICSI). Patients received either Crinone 8% (n = 73) vaginally once daily or two Utrogest capsules (n=53) vaginally three times daily (600 mg). Clinical pregnancy rates were comparable (28.8 versus 18.9%), as were clinical abortion rates until 12 weeks of gestation (14.3 versus 10.0%) and clinical ongoing pregnancy rates (24.7 versus 17.0%) in the Crinone 8% and Utrogest groups, respectively. Forty-seven non-pregnant patients were randomly selected to answer questions regarding comfort during LPS. Crinone 8% had a clear advantage over Utrogest as it resulted in less vaginal discharge (P < 0.01) and fewer application difficulties (P<0.05). Twenty patients familiar with the alternative preparation from a previous cycle also noted that Crinone 8% was easier to apply (P < 0.01) and less time consuming (P < 0.05) to use than Utrogest.

Relationship between sperm DNA damage, induced acrosome reaction and viability in ICSI patients.

The DNA damage in human spermatozoa is a relevant predictor of prognosis in male infertility, whereby increased sperm DNA damage impairs the outcomes of artificial reproduction. Theoretically, DNA damage should alter the special cellular functions of human spermatozoa, and lead to diminished acrosome reaction with reduced fertilization rates. Nevertheless, intracytoplasmic sperm injection (ICSI) has been reported to alleviate such negative outcomes due to DNA damage. This study investigated the relationship between DNA fragmentation and acrosome reaction as well as viability in ICSI patients. The study enrolled 42 men undergoing ICSI due to poor sperm parameters. The DNA fragmentation indexes (DFI) were 4-10% in 38% of the cases, and > or = 10% in 19% of the cases. The results of both acrosome reaction and viability assays showed negative correlations with DFI values in all cases and especially in cases with fertilization rates <60% (P < 0.05). However, such correlations were not found in cases with fertilization rates >60%. There were no live deliveries in patients with high DFI levels (>10%). In conclusion, negative correlations were identified between increased DNA damage, and acrosome reaction and/or viability of human spermatozoa, especially in cases with reduced fertilization rates.