Assisted hatching in mouse embryos using a noncontact Ho:YSGG laser system

Purpose A noncontact holmium:yttrium scandium gallium garnet (Ho:YSGG) laser system has been designed and tested for the micromanipulation of mammalian embryos. The purpose of this preliminary investigation was to determine the effectiveness of this laser for assisted hatching and evaluate its impact on embryo viability. The Ho:YSGG system, utilizing 250-sec pulses at a wave-length of 2.1 m and 4 Hz, was used to remove a portion of the zona pellucida (ZP) of two-to four-cell FVB mouse embryos.Results In the first experiment there was no difference in blastocyst production or hatching rates following laser or conventional assisted hatching (LAH or AH, respectively) in contrast to control embryos cultured in a 5% CO₂ humidified air incubator at 37°C. In the second experiment a blastocyst antihatching culture model was employed and LAH-treated embryos were cultured in a serum-free HTF medium (HTF-o). Blastocyst formation was not influenced by LAH treatment and hatching was increased (P < 0.01) from 4 to 60% compared to HTF-o control group.Conclusions These preliminary data demonstrate the utility and nontoxic properties of the Ho:YSGG laser system for quick and precise ZP drilling.     

Assisted hatching does not enhance IVF success in good-prognosis patients

Purpose: The role of assisted hatching in good-prognosis IVF patients was evaluated in a prospective, randomized, controlled pilot study, which was followed by a retrospective observational series. Methods: After assisted hatching was proved successful in a mouse embryo study, 20 good-prognosis IVF patients were randomly assigned to either assisted hatching (13) or no assisted hatching (7; the controls). Following this series, 27 good-prognosis IVF patients were retrospectively evaluated to determine the outcome with assisted hatching. Results: In the prospective study, clinical pregnancies resulted from 3 (23%) of 13 patients in the hatching group, compared to 3 (43%) of 7 in the control group. Implantation rates were similar: 9.6% in the hatching group and 10.7% in the controls. In the retrospective series, the 11.1% implantation rate with assisted hatching was significantly less than the 42.9% implantation rate seen with traditional IVF. Conclusions: Implantation and pregnancy rates are high in young women undergoing traditional IVF. Assisted hatching is not beneficial in these patients. Presented in part at the 50th Annual Clinical Meeting of the American Society of Reproductive Medicine, Seattle, Washington, October 7–12, 1995.     

Does assisted hatching improve implantation rates after in vitro fertilization or intracytoplasmic sperm injection in all patients? A prospective randomized study

Purpose: Preliminary data from some research centers indicate that assisted hatching might be of value to increase embryo implantation rate in the human, at least in selected cases. It is not clear, however, whether this technique would be of benefit for all patients undergoing an embryo transfer. We therefore performed a prospective randomized study to evaluate the effect of assisted hatching on the implantation rate in our in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) program. Methods: In total, 120 couples undergoing an embryo transfer were randomized between two groups: in one group no assisted hatching was performed (AH–), whereas in the other group the embryos selected for transfer were subjected to partial zona dissection (PZD) immediately prior to the transfer (AH+). Using a computer-generated minimization procedure, patients were allocated to one of the two groups according to four preselected criteria: the number of embryos transferred, the cumulative score of transferred embryos, the age of the patient, and the use of ICSI. Results: Pregnancy and implantation rates in the AH+ and AH– groups were, respectively, 42.1 versus 38.1% and 17.9 versus 17.1%. Conclusions: From our data we conclude that assisted hatching through partial zona dissection prior to embryo transfer does not improve pregnancy and embryo implantation rates in unselected patients undergoing IVF or ICSI.Presented in part at the IXth World Congress on In Vitro Fertilization and Alternate Assisted Reproduction, Vienna, Austria, April 3–7, 1995.     

Enhancing in vitro fertilization of mouse oocytes by partial zona pellucida digestion

This work was undertaken in order to evaluate the effect of partial zona digestion on fertilization in vitro of mouse oocytes and assess zona surface changes induced by the procedure. Three hundred forty-six oocytes allocated for treatment were exposed to Ham's F-10 medium supplemented with 0.5% Pronase for either 3 min (188 oocytes) or 5 min (158 oocytes); 324 oocytes served as controls. Oocyte losses incurred as a result of the procedure were small (15 oocytes; 4.3%). Control and Pronase-treated oocytes were each divided into four subgroups and inseminated with 5 ×10 ⁵,5 ×10 ⁴,5 ×10 ³,or 5 ×10 ² sperm cells/ml. Fertilization was assessed 8 hr following insemination by the appearance of two pronuclei and development to the two- to four-cell stage the following day. The morphology of the zona pellucida following Pronase treatment was assessed by phase-contrast and scanning electron (SEM) microscopies performed immediately after treatment. Fertilization rate of control oocytes was 80% at a sperm concentration of 500,000/ml and gradually declined to ~30% at 500 cells/ml. In contrast, treated oocytes inseminated with 500 sperm cells/ml demonstrated a normal rate of fertilization. At this low sperm concentration the longer Pronase treatment was significantly (P < 0.05) more efficient in enhancing fertilization (69 and 88% for 3 and 5 min of Pronase treatment, respectively). Polyspermic fertilization was not observed in any of the subgroups. Phase-contrast microscopic examination of oocytes at the time of Pronase treatment showed an initial swelling of the zona pellucida for 30–60 sec with a time-dependent increase in its transparency. SEM demonstrated that the fine meshlike structure of the outer surface of zona pellucida digested away, leaving a smoother surface. These morphologic changes were not associated with a diminution in sperm binding or penetration. This work demonstrates that partial zona digestion, which causes uniform dissolution of the zona pellucida and reduction of its thickness, is simple and safe. The procedure significantly increased fertilization efficiency at very low sperm concentrations and could, by itself or in conjunction with other methodologies, improve the reproductive capacity of men producing sperm with a reduced penetrating ability.     

Isolated teratozoospermia and in vitro fertilization

Purpose: To show whether increased amounts of spermatozoa used in insemination of preovulatory oocytes in isolated teratozoospermia according to Kruger's strict criteria gives good fertilization rates. Methods: This study was carried out from September 1993 to November 1994 for a total of 77 cycles, with sperm samples classified according to Kruger's strict classification. Group 1 (C; control) included 37 couples with normal sperm morphology (>14% normal; SC). Group 2 (GP; good prognosis) included 18 couples (morphology, 4 and 14% normal) and group 3 (T; teratozoospermic) included 11 couples, with isolated teratozoospermia in the male partner (morphology, <4% normal). Results: No statistically significant difference was seen in the three groups regarding age, duration of infertility, aspirated follicles, oocytes collected, and embryos transferred. There is a statistically significantly difference (P<0.001) in the number of spermatozoa used in insemination in group 3. Conclusions: In conclusion, a morphological classification using strict criteria in the assessment of sperm morphology is still very important, as increasing the sperm number of spermatozoa inseminated per oocyte may improve zona pellucida binding and give better IVF results.Presented in part at the IXth World Congress on In Vitro Fertilization and Alternate Assisted Reproduction, Vienna, Austria, April 3–7, 1995.     

Zona thinning with a noncontact diode laser in ICSI embryos from women of advanced age

Purpose : The objective of this study was to determine if the zona thinning (ZT) technique improved the rates of implantation and clinical pregnancy for patients aged 38 years submitted to an ICSI program. Methods : A total of 100 patients submitted to ICSI and aged 38 years were divided in a prospective and randomized manner into two groups: Group I – patients submitted to ZT (n = 50); a laser diode with 1.48 m wavelength (Fertilaser) was used for the ZT procedure with 1–2 irradiations of 10 ms applied to four different positions on the zona pellucida (ZP) of each embryo to thin 60–90% of the ZP (each point with a 15–20 m length of ZT). Group II – patients with no ZT (n = 50). In both groups, embryo transfer was performed on the second or third day. Results : The age of Group I patients (39.8 ± 1.3) did not differ (p = 0.67) from that of Group II patients (40 ± 1.9). The number of oocytes retrieved at metaphase II from Group I (6.4 ± 4.2) and Group II (6.8 ± 5) was similar (p = 0.94). Normal fertilization rates and cleavage rates were similar (p = 0.78 and p = 0.63, respectively) for Group I (71.5 ± 22% and 96.7 ± 11%) and Group II (73.5 ± 19.7% and 96 ± 11%, respectively). The number of embryos transferred was similar (p = 0.53) for the two groups (Group I = 3.1 ± 1.3; Group II = 2.9 ± 1.1). The thickness of the ZP of Group I embryos (16.9 ± 2.4 m) did not differ (p = 0.97) from that of Group II embryos (16.9 ± 2.3 m). The rates of embryo implantation and clinical pregnancy per embryo transfer were similar (p = 0.67, p = 0.61) for Group I (7 and 16%, respectively) and for Group II (8.2 and 22%, respectively). Conclusions : These results suggest that ZT in the population aged 38 years may have no impact on ICSI success rates. However, this conclusion is limited to a situation in which length of the laser ZT was 20 m and the laser was applied to four different positions.     

Confounding variables affectingin vitro fertilization success: A decade of experience

Purpose To evaluate etiology of infertility, female age, ovarian response and stimulation protocol in affecting in vitro fertilization (IVF) success.Methods Retrospective analysis was used. Computer records of all 5209 cycles initiated in 2391 couples from February 1, 1984 to December 31, 1993 were analyzed.Results There were 644 intrauterine, 24 solely ectopic and 7 heterotopic pregnancies. Pregnancy rates increased with number of embryos transferred with a significant increase still found between transfer of three or four embryos. Multiple gestations also increased in association with an increasing number of embryos transferred. Pregnancy rates per cycle did not decline with repeated cycles. Reduced pregnancy rates were associated with a primary diagnosis of male infertility. Covariates associated with a better pregnancy rate were younger age of the female partner or higher peak preovulatory serum estradiol. Routine administration of GnRH-agonist was associated with a reduced cycle cancellation rate, an increase in oocytes retrieved and an improved pregnancy rate.Conclusions Couples with male infertility and increased female age should be counselled regarding the reduced prognosis for success.Presented at the 50th Annual Meeting of the American Fertility Society, San Antonio, Texas, November 5–10, 1994.     

Assisted hatching reduces the age-related decline in IVF outcome in women younger than age 43 without increasing miscarriage or monozygotic twinning

Purpose Our purpose was to examine the benefits of assisted hatching in our program and to determine whether the procedure increases the implantation of nonviable embryos or monozygotic twinning. Methods Consecutive in vitro fertilization cycles using assisted hatching were compared with historical controls. The impact of assisted hatching was analyzed according to the woman’s age. Outcome measures were clinical pregnancy, implantation rate, delivery rate, multiple pregnancy, spontaneous abortion, and incidence of monozygotic twins. Results The implantation rate was increased in women aged 35–39 and markedly increased in women aged 40–42. There was no change in spontaneous abortions and no increase in monozygotic twins. Conclusions Assisted hatching is a safe and highly effective adjunct to in vitro fertilization for women aged 35–42 and did not increase spontaneous abortion or monozygotic twinning.     

Successful in vitro fertilization and pregnancy by micromanipulation with epididymal sperm

Purpose To examine the effectiveness of micromanipulation on the treatment of the patients with obstructive azoospermia, subzonal insemination or partial zona dissection was performed using epididymal sperm of the 23 patients, and the in vitro fertilization and embryo transfer (IVF-ET) outcomes were analyzed.Results Fertilized oocytes were obtained in 11 cycles of the 25 treated cycles, and six pregnancies were established by the subsequent ET. Through the analysis of 13 cycles in which both the micromanipulation and usual IVF insemination were performed, the fertilization rate per oocyte with the micromanipulation [25% (28/113)] was significantly higher than that with the usual insemination method [4% (4/102)]. The micromanipulation was most useful for the treatment of the patients with congenital absence of the vas deferens. Moreover, there was no need to aspirate a large volume of the epididymal fluid, but it was essential to prepare more than 10 mature oocytes retrieved.Conclusions These results indicate that micromanipulation with epididymal sperm is an effective treatment for obstructive azoospermia, and careful preparations of sperm and wellcontrolled ovarian hyperstimulation are necessary for successful IVF-ET.     

Comparison of blastocyst transfer to day 3 transfer with assisted hatching in the older patient

OBJECTIVE: To compare cycle outcomes in similar populations of women over 40 who underwent blastocyst transfer compared with women who had day 3 embryo transfer with assisted hatching (ET/AH). DESIGN: Retrospective study. STTING: University hospital-based program. PATIENT(S): Eighty-six IVF cycles in women ages 40 to 43 years who had more than three eight-cell embryos on day 3.On day 3 of embryo culture, patients chose either to undergo blastocyst transfer or day 3 ET/AH. MAIN OUTCOME MEASURE(S): Pregnancy and cryopreservation rates were recorded. RESULT(S): In 48 cycles, blastocyst transfer was performed, and in 38 cycles day 3 ET/AH was performed. There was no statistically significant difference between the blastocyst transfer group and the day 3 ET/AH group with respect to age (41.1 +/- 0.9 years vs. 41.6 +/- 0.8 years), percentage of intracytoplasmic sperm injection cycles (29.2% vs. and 27.6%), number of oocytes (14.9 +/- 5.6 vs. 12.8 +/- 4.0), or number of eight-cell embryos (6.1 +/- 2.2 vs. 5.4 +/- 1.5). Significantly fewers embryos were transferred per cycle with blastocyst transfer (2.6 +/- 1.0) compared with day 3 ET/AH (5.9 +/- 2.0). The viable pregnancy rate was similar in the blastocyst transfer group (29.2%) and in the day 3 ET/AH group (26.3%). Embryos for cryopreservation were available in significantly more cycles in the blastocyst transfer group (52.1%) than in the day 3 ET/AH group (21.1%). Cleavage stage arrest occurred only in one cycle. CONCLUSIONS: Blastocyst transfer appears to be as effective as day 3 ET/AH in older patients with good embryos. Higher cryopreservation rate in the blastocyst transfer group may represent an advantage over day 3 ET/AH. Older women may also benefit from the information that extended culture provides them regarding their oocyte quality.     

In vitro fertilization and gamete micromanipulation in the lowland gorilla

Purpose Our purpose was to utilize current reproductive technologies to treat an infertile female lowland gorilla.Results Following pituitary down-regulation with a gonadotropin releasing hormone agonist and follicular stimulation with human menopausal gonadotropins, transrectal ultrasound-guided aspiration of ovarian follicles yielded nine oocytes. Following failed fertilization with both epididymal and electroejaculated sperm, zona dissection and sperm injection were performed and produced one embryo, which was cryopreserved. Immature oocytes were obtained from ovarian sections and were either cultured in vitro or cryopreserved.Conclusions This report demonstrates that in vitro fertilization techniques, including pituitary suppression, ultrasound-guided aspiration, micromanipulation, and cryopreservation, can be used to assist reproduction in the lowland gorilla.     

Clomiphene citrate and hMG: An alternative stimulation protocol for selected failed in vitro fertilization patients

Purpose This study was carried out to evaluate the potential role of the combination clomiphene citrate/human menopausal gonadotropin (CC/hMG) for patients who failed previous in vitrofertilization (IVF) attempts with gonadotropin-releasing hormone analogs (GnRH- a) and/ or exogenous gonadotropins.Methods Patients were stimulated with CC/hMG (n=93) after unsuccessfully undergoing 182 gonadotropin cycles with (n=106) or without (n=76) luteal-phase GnRH- a. Cancellation rate, length of stimulation, and peak estradiol levels did not differ significantly between the two regimens.Results Although fewer oocytes were retrieved when the CC/hMG combination was used, 16 patients were able to successfully achieve a pregnancy (26.2% delivery rate/ transfer). When daily follicle stimulating hormone (FSH) levels were measured in two successive cycles in those women who conceived with the CC/hMG stimulation, baseline levels did not differ when compared with a previous GnRH-a/hMG cycle. Nevertheless, serum FSH levels rose rapidly and remained higher in the GnRH- a/hMG cycle, reaching significantly higher levels on day of human chorionic gonadotropin (hCG) administration.Conclusion Selected patients who failed previous IVF attempts with gonadotropins with or without GnRH analogs may benefit from the addition of CC to their ovarian stimulation protocol.Presented at the 50th Annual Meeting of the American Fertility Society, San Antonio, Texas, November 5–10, 1994.     

The predictive value of serum progesterone and 17-OH progesterone levels onin vitro fertilization outcome

Purpose In order to identify parameters which predict prognosis for success with in vitro fertilization, 17-hydroxyprogesterone and progesterone levels were evaluated in 254 patients undergoing 296 in vitro fertilization cycles. Selected response and outcome data were recorded.Results Patients with intermediate values of serum progesterone (0.7–0.8 ng/ml) at the time of human chorionic gonadotropin administration achieved significantly higher pregnancy rates than patients with lower (<0.7 ng/ml) or higher (>0.8 ng/ml) levels. The clinical pregnancy rates were 46%, 31%, and 27% respectively (P = 0.02). There was no change in 17-hydroxyprogesterone concentration which predicted a higher pregnancy rate.Conclusion Excellent clinical pregnancy rates were noted in cycles with a progesterone level of 0.7–0.8 ng/ml, as well as good results in cycles above 0.8 ng/ml. There is therefore no reason to administer human chorionic gonadotropin at a smaller follicle size to prevent a rise in serum progesterone.Presented at the 50th Annual Meeting of the American Fertility Society, November 5–10, 1994, San Antonio, Texas.     

Blocking of human fertilization in vitro by sera with sperm-immobilizing antibodies

Serum samples with sperm-immobilizing antibody activity from six women were examined for ability to block sperm-egg interaction by a zona penetration test where human follicular ova matured in vitro were used. Exposure of spermatozoa from a fertile healthy donor to the sera impaired binding to and penetration through the zona pellucida of the spermatozoa completely in five cases and incompletely in one case. Successful fertilization in vitro was achieved by using fetal cord serum instead of autoserum of the patient included in the in vitro fertilization and embryo transfer program. These results suggest that interference with sperm-egg interaction may be an additional mechanism of infertility that is caused by antisperm antibodies.     

In Vitro fertilization: A cost-effective alternative for infertile couples?

Objective To evaluate the cost ofin vitro fertilization by calculating the cost of a live birth using this technology and determine cost variation according to the clinical characteristics of a particular population.Design Retrospective review of infertile couples who presented for their first IVF cycle in 1993. A fraction of the total population was assigned to three groups A, B, and C with high, intermediate and low probability of pregnancy respectively and their reproductive performance was evaluated until September 1994 or a maximum of three IVF cycles have been completed.Setting Thein vitro fertilization program at the Brigham and Women's Hospital, Boston.Patients 182 couples who presented for their first IVF cycle in 1993.Main Outcome Measure The cost of a successful pregnancy using IVF in the three groups and in the general population was calculated by dividing the average cost of an IVF cycle by the fraction of the cycles resulting in a successful pregnancy.Results The cost of a successful pregnancy in group A, B and C ranged from $22,857 to $42,666 after 1 cycle and from $26,800 to $74,666 after 3 IVF cycles. The average cost for the 182 patients was $29,120 after 1 cycle and $31,590 after a maximum of 3 IVF cycles.Conclusion The cost of a successful pregnancy: (1) was comparable to other options available to an infertile couple such as adoption and tubal surgery, (2) was 50% to 70% cheaper in the group with a highest probability of pregnancy when compared to the group with the lowest probability of pregnancy, and (3) did not vary significantly after 1 or 3 IVF cycles in most groups.