In Vitro Microvibration Increases Implantation Rate After Embryonic Cell Transplantation.

In natural conditions the oocyte and embryo are subjected to ever-changing dynamic processes. However, the routine assisted reproductive technologies today involve the use of static in vitro culture systems. The objective was to determine whether there is any difference in the viability of embryos after in vitro culture under static and mechanical microvibration conditions. The viability of embryonic cells (9,624 embryos) generated from 4,436 couples after in vitro culture was evaluated. For groups ≤29, 30-34, 35-39, and ≥40 years, the following rates of high-quality embryos without fragmentation (two to four blastomeres on day 2; six to eight blastomeres and compacting morula on day 3; blastocyst, expanded and hatching blastocyst on day 5) were detected (static vs. vibration, respectively): 65% versus 71%, 44% versus 69%, 67% versus 76% (for statistically significant differences between respective rates in these three groups, p < 0.05), and 67% versus 66% (p > 0.1). The following baby-take-home rates were determined for groups ≤29, 30-34, 35-39, and ≥40 years (static vs. vibration, respectively): 30% versus 31% (p > 0.1, increasing only on the level of tendency), 28% versus 37%, 23% versus 29%, and 9% versus 15% (differences between respective rates in these three groups with p < 0.05). It was concluded that in vitro culture of embryos under microvibration (with a mimic of conditions in nature whereby oviductal fluid is mechanically agitated by the epithelial cilia) significantly increases the baby-take-home rate for patients 30 years and older.

"Naturalization" of Routine Assisted Reproductive Technologies by In Vitro Culture of Embryos with Microvibration: Sex Ratio, Body Length, and Weight of 2,456 Live-Birth Deliveries after Transfer of 9,624 Embryos In Vitro Cultured in Static System and with Microvibration.

Aim was to determine whether there is any difference in the sex ratio, body length, and body weight of 2,456 deliveries after transfer of 9,624 embryos derived using in vitro culture under static and mechanical microvibration conditions. Pronuclear embryos from 4435 patients were cultured in vitro under two different conditions: without (n = 4821) and with mechanical agitation (n = 4803). Sex ratio, body length, and weight of 2,456 live-birth deliveries after transfer of 9,624 embryos were noted. The proportion of males at birth was significantly associated with mode of in vitro culture of embryos only among women aged 40 years and older. The rate "body length" was significantly associated with mode of in vitro culture of embryos only among women aged 29 and younger. In the same time, among twins, this ratio positively associated with in vitro culture of embryos under microvibration only among women aged 30-34 years as well as ≥40 years and negatively among women aged 35-39 years. It was concluded that birth weight of infants was positively associated with mode of in vitro culture of embryos under microvibration among women of all age groups. This trial registration number is ISRCTN13773904, registered 6 April 2016.

In-vitro culture of human embryos with mechanical micro-vibration increases implantation rates.

The in-vitro culture of human embryos in a medium subjected to regular short intervals of mechanical agitation leads to increased development rates. This type of treatment tries to mimic conditions in nature whereby oviductal fluid is mechanically agitated by the epithelial cilia. This phenomenon can be explained by the fact that an embryo developing in vivo is naturally exposed to constant vibrations of around 6Hz with the periodically repeating increase to 20Hz. This review covers the history of this question and in this light offers an explanation through biological concept for one of the most recent developments in this area: in-vitro culture of human embryos with mechanical micro-vibration. The effect of mechanical micro-vibration on embryos during their in-vitro culture was examined. Pregnancy rates after the transfer of embryos in the group with in-vitro culture under mechanical vibration were increased.

Mechanical agitation during the in vitro culture of human pre-implantation embryos drastically increases the pregnancy rate.

BACKGROUND: The in vivo developing embryo is naturally exposed to constant vibrations of around 6 Hz, increasing to 20 Hz when the oviductal fluid is mechanically agitated by the cilia. This study examines the effects on viability of subjecting human pronuclear oocytes and embryos to mechanical agitation during their in vitro culture before transplantation. METHODS: Metaphase-II oocytes were ICSI/IVF with morphologically normal spermatozoa and then divided into two groups according to whether the cells underwent mechanical agitation (20 Hz over 5 seconds once per hour) of the culture medium (Group 2, n=23) or were cultured without mechanical agitation (Group 1, n=23). The fertilization rate of oocytes was recorded 18 hours later. Embryo development was monitored every day during the whole period of in vitro culture up to the embryo transfer on day 3, 4 or 5. RESULTS: Pregnancy rates after the transfer of 3 Day embryos in Group 1 and Group 2 were 50% and 80%, and of 5 Day embryos in Group 1 and Group 2 were 36% and 73%, respectively. CONCLUSIONS: The in vitro culture of human embryos in a medium subjected to regular short bursts of mechanical agitation drastically increases their development rate.

Pregnancy after the calcium ionophore correction of pronuclei position in oocytes after intracytoplasmic sperm injection.

OBJECTIVE: To report a successful pregnancy after transfer of embryos derived from oocytes after calcium ionophore correction of the pronuclei localization after intracytoplasmic sperm injection (ICSI). DESIGN: Case report. SETTING: University hospital. PATIENT(S): A 30-year-old patient and her 30-year-old husband, diagnosed with asthenoteratozoopermia, underwent ICSI because of three unsuccessful IUI attempts. INTERVENTION(S): Thirteen metaphase II oocytes were injected with morphologically normal spermatozoa and immediately divided into two groups. Group 1 (n = 7) was the untreated control and in group 2 (n = 6), the oocytes were treated with 10 µM calcium ionophore solution for 20 minutes at 37°C in 6% CO(2). The fertilization was checked 18 hours later and location of pronuclei in cytoplasm was assessed. Transfer of two embryos was performed on the third day after oocyte retrieval. MAIN OUTCOME MEASURE(S): Pregnancy after transfer of embryos after calcium ionophore correction of pronuclei localization. RESULT(S): Fertilized zygotes from calcium ionophore-treated oocytes with physiologically normal central localization of pronuclei were chosen for ET. Clinical pregnancy was confirmed at 7 weeks of gestation. CONCLUSION(S): Post-ICSI calcium ionophore activation of oocytes can be used for correction of the pronuclei localization, which can increase developmental rate of embryos.

Rescue of spermatogenesis arrest in azoospermic men after long-term gonadotropin treatment.

Several studies have shown that FSH treatment can improve sperm production quantitatively and increase the spermatogonial population in oligozoospermic men with normal hormonal profiles. In this study, we describe the results of long-term gonadotropin therapy of normogonadotropic patients with nonobstructive azoospermia.

Quantitative evaluation of spermatozoa ultrastructure after acupuncture treatment for idiopathic male infertility.

OBJECTIVE: To evaluate the ultramorphologic sperm features of idiopathic infertile men after acupuncture therapy. DESIGN: Prospective controlled study. SETTING: Christian-Lauritzen-Institut, Ulm, IVF center Munich, Germany, and Department of General Biology, University of Siena, Siena, Italy. PATIENT(S): Forty men with idiopathic oligospermia, asthenospermia, or teratozoospermia. INTERVENTION(S): Twenty eight of the patients received acupuncture twice a week over a period of 5 weeks. The samples from the treatment group were randomized with semen samples from the 12 men in the untreated control group. MAIN OUTCOME MEASURE(S): Quantitative analysis by transmission electron microscopy (TEM) was used to evaluate the samples, using the mathematical formula based on submicroscopic characteristics. RESULT(S): Statistical evaluation of the TEM data showed a statistically significant increase after acupuncture in the percentage and number of sperm without ultrastructural defects in the total ejaculates. A statistically significant improvement was detected in acrosome position and shape, nuclear shape, axonemal pattern and shape, and accessory fibers of sperm organelles. However, specific sperm pathologies in the form of apoptosis, immaturity, and necrosis showed no statistically significant changes between the control and treatment groups before and after treatment. CONCLUSION(S): The treatment of idiopathic male infertility could benefit from employing acupuncture. A general improvement of sperm quality, specifically in the ultrastructural integrity of spermatozoa, was seen after acupuncture, although we did not identify specific sperm pathologies that could be particularly sensitive to this therapy.

Higher aneuploidy rates of chromosomes 13, 16, and 21 in a patient with globozoospermia.

OBJECTIVE: To analyze rates of chromosomal anomalies in a patient with globozoospermia. DESIGN: Case report. SETTING: Tertiary-care infertility clinic. PATIENT(S): A 44-year-old man with 100% globozoospermia. INTERVENTION(S): Fixation of sperms and analysis of aneuploidies and diploidies by fluorescence in situ hybridization. MAIN OUTCOME MEASURE(S): Aneuploidy rates of chromosomes 13, 16, and 21 and diploidy rate in a patient with 100% globozoospermia. RESULT(S): The aneuploidy rates in spermatozoa from a patient with globozoospermia were significantly higher than in a normospermic man. The incidence of a disomy of the chromosomes 13, 16, and 21 was 4%, 5%, and 1%, respectively, and the nullisomy rates were 2%, 2%, and 3%, respectively. CONCLUSION(S): This study demonstrates a positive correlation between globozoospermia and higher chromosomal aneuploidies of the chromosomes 13, 16, and 21, suggesting a higher risk of creating aneuploid embryos after intracytoplasmic sperm injection treatment for globozoospermia patients.

Gonadotropin treatment of an azoospermic patient with a Y-chromosome microdeletion.

OBJECTIVE: To report on the treatment with recombinant FSH of an azoospermic normogonadotropic patient with a Y-chromosome microdeletion. DESIGN: Case report. SETTING: An assisted reproduction center. PATIENT(S): A 32-year-old patient with azoospermia. INTERVENTION(S): Histological testicular biopsy, Y-chromosome screening, FSH treatment, and intracytoplasmic sperm injection. MAIN OUTCOME MEASURE(S): Fertilization, embryo development, pregnancy, and delivery. RESULT(S): After 6 months of treatment with gonadotropins, a small number of spermatozoa were found in the patient's ejaculate and used for IVF, resulting in a successful twin pregnancy and the delivery of two healthy girls. CONCLUSION(S): In this study, treatment with gonadotropins promoted the spermatogenesis process and led to the production of spermatozoa in a normogonadotropic azoospermic patient.

Improved implantation rate after chemical removal of the zona pellucida.

OBJECTIVE: To evaluate the implantation rate achieved after chemical removal of the zona pellucida from day 5 human in vitro-derived embryos. DESIGN: Prospective, randomized, controlled study. SETTING: A tertiary care infertility clinic. PATIENT(S): Two hundred fifty-seven patients undergoing IVF with transfer of morulas or blastocysts on day 5 after oocyte retrieval. All patients had had at least two previous implantation failures. INTERVENTION(S): Chemical removal of zona pellucida by using acidic Tyrode's solution vs. no removal (controls). MAIN OUTCOME MEASURES: Clinical pregnancy rate and implantation rate per transfer. RESULT(S): Embryos without zona pellucida implanted at nearly twice the rate of control embryos (15.7% vs. 27.5%). The pregnancy rate was also significantly higher in the zona pellucida-free group than the control group (31.0% vs. 46.1%). Removal of zona pellucida was most effective in embryos with delayed development, which reached the morula or early cavitating stage on day 5 of in vitro culture (implantation rate, 12.1% vs. 25.7%). CONCLUSION(S): Chemical removal of zona pellucida from day 5 in vitro cultured human embryos is an effective and safe method of significantly improving the implantation rate, especially of embryos with delayed development.

Effect of highly purified urinary follicle-stimulating hormone on oocyte and embryo quality.

OBJECTIVE: To determine the effects of ovarian stimulation with highly purified urofollitropin on oocyte and embryo quality. DESIGN: Parallel randomized open-label clinical study. SETTING: Assisted reproduction centers. PATIENT(S): Two hundred sixty-seven infertile couples undergoing IVF/ICSI. INTERVENTION(S): All participants underwent standard down-regulation with GnRH analogue. One hundred thirty-three participants received highly purified urinary FSH and 134 controls received recombinant FSH. MAIN OUTCOME MEASURE(S): Primary end points were number of morphologically mature oocytes retrieved, embryo quality, and pregnancy and implantation rates. Secondary end points were: total number of days of FSH stimulation, total dose of gonadotropin administered, fertilization rate per number of retrieved oocytes, embryo cleavage rate, live birth and miscarriage rates, endometrial thickness and estradiol level on the day of hCG administration, cancellation rate, and incidence of moderate or severe ovarian hyperstimulation syndrome. RESULT(S): Pregnancy and implantation rates were nonsignificantly higher in the urinary FSH group than the recombinant FSH group (46.5% vs. 36.8% and 22.1% vs. 15.8%, respectively). The grade 1 embryo score was significantly higher in the urinary FSH group than the recombinant FSH (42.1% vs. 33.5%), and the live birth rate was nonsignificantly higher in the former group. CONCLUSION(S): Highly purified urinary FSH is as effective, efficient, and safe for clinical use as recombinant FSH.

Influence of acupuncture on the pregnancy rate in patients who undergo assisted reproduction therapy.

OBJECTIVE: To evaluate the effect of acupuncture on the pregnancy rate in assisted reproduction therapy (ART) by comparing a group of patients receiving acupuncture treatment shortly before and after embryo transfer with a control group receiving no acupuncture. DESIGN: Prospective randomized study. SETTING: Fertility center. PATIENT(S): After giving informed consent, 160 patients who were undergoing ART and who had good quality embryos were divided into the following two groups through random selection: embryo transfer with acupuncture (n = 80) and embryo transfer without acupuncture (n = 80). INTERVENTION(S): Acupuncture was performed in 80 patients 25 minutes before and after embryo transfer. In the control group, embryos were transferred without any supportive therapy. MAIN OUTCOME MEASURE(S): Clinical pregnancy was defined as the presence of a fetal sac during an ultrasound examination 6 weeks after embryo transfer. RESULT(S): Clinical pregnancies were documented in 34 of 80 patients (42.5%) in the acupuncture group, whereas pregnancy rate was only 26.3% (21 out of 80 patients) in the control group. CONCLUSION(S): Acupuncture seems to be a useful tool for improving pregnancy rate after ART.

Twin pregnancy after vitrification of 2-pronuclei human embryos.

OBJECTIVE: To report an ongoing twin pregnancy after transfer of embryos that were vitrified at the 2-pronuclei stage in a new vitrification solution. DESIGN: Case report. SETTING: A tertiary-care infertility clinic. PATIENT(S): A 26-year-old infertile woman in whom two previous IVF implantations failed. INTERVENTION(S): Vitrification of 2-pronuclei embryos, in vitro culture for 48 hours, and transfer into the uterus. MAIN OUTCOME MEASURE(S): Survival and cleavage after vitrification and achievement of clinical pregnancy. RESULT(S): Six zygotes were vitrified by using a three-step protocol (4% ethylene glycol for 3 minutes, 20% ethylene glycol for 1 minute, and 38% ethylene glycol and 1.2 M trehalose for 0.5 minute). After 2 months of storage in a double-straw system in liquid nitrogen, two zygotes were warmed and cryoprotectants were removed by using a four-step protocol (1 M, 0.5 M, 0.25 M, and 0.125 M of trehalose). Two embryos were transferred after 48 hours of in vitro culture, cleaving to 5 and 6 cells. The resulting twin pregnancy was confirmed by ultrasonography at the sixth week. CONCLUSION(S): Vitrification by using ethylene glycol and trehalose appears to be a safe, promising method for cryopreservation of human zygotes. Storage of vitrified zygotes in a double-straw system does not compromise their subsequent potential for survival and development.