A comparative analysis of embryos derived from routine in-vitro fertilization and subzonal microinsemination.

Embryos obtained from patients undergoing routine in-vitro fertilization (IVF) and embryo transfer were compared with those undergoing subzonal microinsemination (SUZI) for male factor infertility. Overall, the proportion of cleaved embryos was significantly higher in the IVF group in comparison with the SUZI group at 48 h post-insemination [1533 out of 1609 (95.3%) versus 776 out of 952 (81.5%)]. The mean +/- SD grading score of the IVF-derived embryos of 3.61 +/- 0.50 was significantly better than that for SUZI of 2.97 +/- 0.86 (P < 0.0005) at the same time. The implantation rates following the replacement of IVF or SUZI embryos at 48 h were comparable: 14.3 and 10.0% respectively. However, the IVF embryo implantation rate of 15.1% at 72 h was significantly better than that following the replacement of SUZI embryos at either 48 (10.0%) or 72 h (8.0%). The replacement of SUZI-derived embryos at 48 h resulted in significantly higher pregnancy (25.0%) and implantation rates (10.0%) than at 72 h, with rates of 10.8 and 8.0% respectively. Similarly, the overall embryo quality deteriorated following in-vitro culture for up to 72 h. The clinical pregnancy loss rate (33.0%) was highest following the replacement of SUZI embryos at 72 h, although the data were limited. It is suggested that these data indicate that a combination of in-vitro manipulation, the injection of multiple spermatozoa into the subzonal space and probably the genomic capacity of spermatozoa derived from poor-quality semen may contribute to the poorer outcome of embryo development following SUZI. Prolonged in-vitro culture beyond 48 h appears to be deleterious to the development of SUZI cleaved embryos and the subsequent outcome of treatment, and hence should be avoided.

Clinical experience with repeat subzonal microinsemination of oocytes failing to fertilize after an initial microinsemination.

We report on our experience with repeat subzonal microinsemination of oocytes with spermatozoa after the failure of fertilization after an initial procedure. The data indicate that the practice contributed positively toward the overall outcome of treatment. In our view, this practice needs to be incorporated into existing micromanipulation programs.

Absence of heat treatment of serum for culture medium supplementation does not adversely affect the outcome of in-vitro fertilization.

This study was carried out to determine if not heat-treating serum prior to use for medium supplementation adversely affected in-vitro fertilization (IVF) of human oocytes. Morphologically mature human oocytes derived from 135 patients undergoing IVF treatment were studied. A total of 504 oocytes were incubated, inseminated and the resulting pronuclear oocytes cultured further in Earle's balanced salt solution (EBSS) supplemented with 10% non-heat-treated serum. Comparisons of fertilization rate and embryonic development were made between these and 687 control oocytes derived from the same patients but incubated, inseminated and resulting pronuclear oocytes cultured further in EBSS supplemented with 10% heat-treated serum. The fertilization rate of 74.4% (375/504) of oocytes handled in serum-supplemented medium that had not been heat-treated was significantly better than the rate of 67.7% (465/687) for controls (P < 0.0125). The proportion of pronucleate oocytes that cleaved was also significantly better in the non-heat-treated serum group: 270/300 (90%) versus 307/375 (81.8%) (P < 0.0025). There was no significant difference in the proportion of embryos with four or more cells at the time of embryo transfer. The results show that the absence of heat treatment of serum used to supplement culture medium has no adverse effect on the fertilization rate and short-term embryo development in vitro; hence we suggest that serum heat treatment is an unnecessary procedure and could be abandoned.

The influence of subzonal microinsemination of oocytes failing to fertilize in scheduled routine in-vitro fertilization cycles.

The subzonal microinsemination (SUZI) of oocytes failing to fertilize following routine in-vitro fertilization (IVF) insemination was studied in comparison to routine reinsemination. The data presented indicate that fertilization rate was significantly better with SUZI (23.4%) than with reinsemination (8.3%). The percentage of cleaved replaceable embryos generated was also significantly higher following SUZI than with reinsemination (96.6% versus 66.7% respectively). SUZI led to a significantly higher increase in the percentage of additional number of fertilized oocytes (100%), fertilization cycles (185%), embryos replaced (108%) and embryo replacement cycle over that generated by initial insemination. These were in comparison to 26, 14.3, 11.6 and 15%, respectively, as a result of reinsemination. No pregnancy occurred following replacement of embryos generated from reinsemination. Six pregnancies resulted from replacement of embryos following SUZI. We suggest therefore that SUZI should be the preferred option of practice when oocytes have failed to fertilize following initial insemination for routine IVF.

In vitro fertilization and embryonic development of oocytes fertilized by sperm treated with 2-deoxyadenosine.

OBJECTIVE: To test the effect in an assisted pregnancy program of an agent known to enhance sperm motility and oocyte penetration ability. DESIGN: Prospective with internal control. SETTING: Hospital clinic. PATIENTS AND INTERVENTIONS: Forty-two oocytes obtained from women undergoing IVF were inseminated with their husband's sperm washed, incubated, and capacitated in Earle's medium supplemented with 1 mM, 2.5 mM, or 5 mM 2-deoxyadenosine (2-DXA). The outcome of insemination was compared with those of 234 control oocytes from the same women that were inseminated with husband's sperm washed in 2-DXA-free medium. 15, 14, and 13 oocytes were inseminated with sperm washed in 1 mM, 2.5 mM, and 5 mM 2-DXA, respectively. RESULTS: Fertilization rates in the respective groups were 93%, 85.7%, and 85.5%. These were higher than the 70.1% fertilization rate in the control group, but only statistically higher (P < .002) in the 1 mM 2-DXA group. Embryonic development in all three 2-DXA groups was comparable to the controls. CONCLUSION: It is suggested that the value of sperm motility enhancing agents requires further evaluation in assisted pregnancy programs.

Successful twin pregnancy and delivery after microinseminated oocyte fallopian transfer for male factor infertility.

The case described indicate that microinseminated oocyte fallopian transfer may be a potentially useful option of management of male factor infertility. The question of the effect of a breach in the oocyte/embryo zona during micromanipulation procedure on the outcome of their in vitro culture remains to be addressed.

Subzonal multiple sperm injection in the treatment of previous failed human in vitro fertilization.

OBJECTIVE: To assess the value of subzonal sperm injection in previous failed spontaneous in vitro fertilization (IVF). DESIGN: Prospective study on patients with at least one cycle of previous failed spontaneous IVF. SETTING: Human Reproductive Biology Unit, Soliman Fakeeh Hospital, Jeddah, Saudi Arabia. PATIENTS: Thirty-nine patients with prolonged infertility with at least one previous cycle of failure of spontaneous fertilization of any oocyte. RESULTS: Seventy-eight of 276 oocytes successfully injected with three to four sperms were fertilized. Fertilized rate was 28.2%. Polyspermy was observed in 6 (8%) of the fertilized oocytes. Sixty-six (92%) of the 72 with monospermic fertilization cleaved normally and were replaced in the uterine cavity. Pregnancy occurred in 6 (23%) of the women who had embryos replaced. CONCLUSIONS: Multiple sperm injection into the oocyte subzonal space should be considered for the treatment of patients with previous failed spontaneous IVF.

Changes in follicular fluid gas and pH during carbon dioxide pneumoperitoneum for laparoscopic aspiration and their effect on human oocyte fertilizability.

OBJECTIVE: To determine changes in follicular fluid (FF) gases and acidity (pH) during either ultrasound (US)-guided or laparoscopic aspiration under carbon dioxide (CO2) pneumoperitoneum and relate the findings to the outcome of oocyte fertilization in vitro. DESIGN: A prospective study carried out over a limited period of 1 month during which test FF samples were obtained without direct exposure to CO2 or air. SETTING: Within the assisted pregnancy program setting at the Human Reproductive Biology Unit at the Soliman Fakeeh Hospital. PATIENTS: Infertile women undergoing either laparoscopic or US guided follicular aspiration for the purpose of in vitro fertilization treatment. INTERVENTION: Laparoscopic follicular aspiration under 100% CO2 pneumoperitoneum or transurethral US-guided follicular aspiration. RESULTS: Mean FF carbon dioxide partial pressure (pCO2) and oxygen partial pressure (pO2) were significantly higher in those aspirated at laparoscopy compared with those obtained by US-directed aspiration. The mean FF pH in the laparoscopic group was as a consequence significantly lower than in the US group. Oocyte fertilizability was significantly reduced in the laparoscopic aspiration group compared with the US group. This was more pronounced when the fertilization rate of the last recovered oocytes from the groups were compared. No difference was observed in the post fertilization embryonic development between the two groups. CONCLUSION: One hundred percent pneumoperitoneum leads to changes in FF that adversely affect oocyte fertilizability in a time-dependent manner. The use of US-directed procedure is advocated for all patients undergoing follicular aspiration in assisted pregnancy program. Where this is not feasible, the duration of CO2 pneumoperitoneum should be minimized.

Fertilization and embryonic development of human oocytes after cooling.

Injury to living cells resulting from rapid cooling to temperatures at or near 0 degrees C has long been recognized, and the phenomenon, which is termed 'cold shock', has been known to occur in some mammalian gametes. Although human embryos have been successfully stored at low temperatures, cryopreservation of the human oocyte is proving to be more difficult. Whether or not this lack of success is a direct result of cellular injury brought about by 'cold shock' is the purpose of the current investigation. Human oocytes were cooled, in the absence of cryoprotectants, at two different cooling rates (-3 degrees C/min and -1000+ degrees C/min) to a temperature of 0 degrees C and rewarmed prior to insemination. In both cases fertilization after cooling was similar to the rates achieved in a routine in-vitro fertilization and embryo transfer procedure. After cooling at -3 degrees C/min, the rate of fertilization was 19/22 (86%) and after cooling at -1000+ degrees C/min, 9/9 (100%), with non-cooled control rates of 62/87 (71%) and 35/50 (70%) respectively. Fertilized oocytes from both groups were successfully cultured for a further 24 h before termination of the experiment.

Survival of human oocytes cryopreserved with or without the cumulus in 1,2-propanediol.

BACKGROUND: Although cryopreservation of human preembryos has been carried out with success, the cryostorage of oocytes, which pose fewer controversial moral, ethical, and legal problems has been much less successful. Various attempts to cryopreserve human oocytes have been mostly unsuccessful and the search for an optimal protocol for oocyte cryopreservation remains elusive. We therefore undertook this study to determine the effect of oocyte cryostorage in 1,2-propanediol. METHOD: Mature human oocytes with or without their cumuli were cryopreserved in precooled 1,2-propanediol, then thawed and inseminated with sperms for in vitro fertilization. The outcome of insemination and subsequent embryonic development were also recorded and compared. RESULTS: Postthaw cryosurvival rate was significantly better when cryostorage was carried out with the oocyte cumulus intact as compared to those oocytes denuded of their cumuli (54 versus 27%, respectively; P < 0.05). Eight (44%) of 18 surviving postthaw oocytes with intact cumuli were fertilized normally, with cleavage in six, as compared to two (25%) and one, respectively, of those denuded of their cumulus prior to cryostorage. Development to the blastocyst stage was achieved in three embryos derived from oocytes with an intact cumulus at cryostorage. CONCLUSION: We conclude that 1,2-propanediol can be used with success in oocyte cryopreservation, although the issue of parthenogenecity is still to be resolved. Oocyte's with intact cumulus survive cryostorage better than those without it.

The effect of caffeine on the ability of spermatozoa to fertilize mature human oocytes.

Although caffeine has been reported to enhance spermatozoon motility as well as fertilizing ability, its use in clinical practice has remained sparse. We report here the results carried out to assess the effect of exposing normal human spermatozoa to different concentrations of caffeine on their motility, their ability to fertilize oocytes, and the subsequent development of resulting embryos. Mature human oocytes were inseminated with spermatozoa washed and capacitated in 1.25, 2.5, and 5 mM caffeine. The fertilization rates were compared with control oocytes inseminated with untreated spermatozoa. While caffeine was observed to improve significantly various motility parameters in a dose-dependent manner, it did not lead to an improvement in the fertilization rates. At the highest concentration, 5 mM, it adversely affected the fertilization rate: 38%, compared with 78% in controls. Embryonic development was also observed to be retarded at the lower concentrations, while it was virtually inhibited in the 5 mM concentration group. Our results suggest that while a definite improvement in motility may occur when spermatozoa are exposed to caffeine, this improvement did not translate into enhanced fertilizing ability and subsequent embryonic development. We are therefore of the opinion that the use of caffeine as a spermatozoon motility enhancer requires further studies prior to wider clinical use in assisted pregnancy programs.

Successful use of the sperm motility enhancer 2-deoxyadenosine in previously failed human in vitro fertilization.

The outcome of in vitro fertilization treatment in male-factor infertility is generally poor, due mainly to poor fertilization rate and hence fewer available embryos for replacement. This study was carried out to assess the value of asthenospermic sperm exposure to a motility enhancing agent, 2-deoxyadenosine (2-DXA), on our in vitro fertilization program in couples undergoing repeat treatment after previous failed fertilization. Following sperm wash and incubation in 2-DXA-supplemented medium, marked significant improvements were observed in the sperm motility pattern and the number of recoverable sperms as compared to control unexposed samples. There was also a significantly better fertilization rate and higher number of replaceable embryos available following sperm wash in 2-DXA as compared to those washed without it. Following embryo transfer, pregnancy rate was comparable to the generally reported pregnancy rates for routine in vitro fertilization and embryo transfer treatment. Three normal babies were born. Our results indicate that 2-DXA enhances fertilization rate in some asthenospermic patients and that it has no adverse effect on embryonic development.

A new approach to the management of patients at risk of ovarian hyperstimulation in an in-vitro fertilization programme.

In this study, we report an alternative method of management of patients at a potential risk of ovarian hyperstimulation syndrome (OHSS) in an in-vitro fertilization (IVF) programme, by the use of gonadotrophin releasing hormone analogue (GnRHa). Thirty eight women considered at risk of this syndrome, having serum oestradiol greater than 4000 pg/ml following ovarian stimulation, received a GnRHa nasal spray for induction of the preovulatory endogenous luteinizing hormone surge for follicular maturation prior to oocyte recovery. Oocyte recovery (mean oocytes per cycle 18.60 +/- 4.79; range 15-35) and IVF were successfully completed in 27 cycles. Twenty six women had embryos replaced and 11 pregnancies occurred (28.9% per operation, 42.3% per replacement cycle). None of the patients developed OHSS. Where there is a risk of OHSS, the use of GnRHa to induce the preovulatory surge of endogenous luteinizing hormone for final follicular maturation provides a successful and more economical alternative to cancellation of cycles.

Stimulation of endogenous surge of luteinizing hormone with gonadotropin-releasing hormone analog after ovarian stimulation for in vitro fertilization.

The effect of induction of preovulatory endogenous surge of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) with intranasal administration of GnRH-analog (GnRH-a) in an in vitro fertilization (IVF) program is reported. The use of GnRH-a resulted in a significantly better percentage of replaceable embryos (91% versus 85%). The pregnancy rate was 51% in comparison with 32% in control cycles in which follicular maturation was achieved by human chorionic gonadotropin administration. There was no significant difference in the postoocyte recovery serum progesterone patterns between the two groups. Our results indicate that the induction of endogenous LH and FSH surge with GnRH-a may be successfully employed for final follicular maturation after ovarian suprastimulation without affecting the outcome of IVF adversely. Apart from being a more physiological approach to oocyte maturation, it also has potential economic and clinical advantages.

Osmotic response of oocytes using a microscope diffusion chamber: a preliminary study comparing murine and human ova.

The hydraulic conductivity (Lp) of the plasma membrane determines how cells respond to the stresses of dehydration encountered during cryopreservation. We have used a microscope diffusion chamber which allows for direct real-time observation of the dynamic osmotic response of individual cells in microvolume suspension to compare the Lp of murine and human unfertilized ova. In this system, the response of an individual cell to the induced osmotic imbalance is documented via a series of photomicrographs or videotape; from these data the Lp can be computed. Donated human preovulatory oocytes were compared with macroscopically normal human ova, 43 hr after insemination, which had failed to fertilize (Ff) and with murine ova collected 13 hr post human chorionic gonadotropin injection. The permeability coefficients were 0.65 +/- 0.43, 0.84 +/- 0.39, and 0.36 +/- 0.07 micron3/micron2/atm/min. The results suggest that it may be possible to use Ff ova for experiments to design suitable cryopreservation procedures.

In vitro fertilization in women with "frozen pelvis": clinical outcome of treatment.

The outcome of 93 consecutive cycles of in vitro fertilization (IVF) and embryo transfer (ET) treatment in 52 women with "frozen pelvis" is reported. The treatment termination rate due to inadequate ovarian stimulation was 20%. The mean +/- standard deviation of the number of follicles of mean diameter greater than or equal to 15 mm, oocytes recovered and fertilized per operation, and embryos replaced per cycle were 4.80 +/- 2.07, 4.02 +/- 2.32, 2.54 +/- 2.06, and 2.57 +/- 1.30, respectively. The pregnancy rate per ET was 29.6%. These data compare with those of other treated women with adhesion-free ovaries over the same period, suggesting that pelvic adhesive disease has no adverse effect on outcome of IVF-ET.

Ultrasound measurement of endometrial thickness on different ovarian stimulation regimens during in-vitro fertilization.

Endometrial thickness was measured ultrasonographically in three groups of patients undergoing in-vitro fertilization (IVF) on three different ovulation induction regimens. The endometrial thickness was comparable on all three regimens and similar to that observed in a group of spontaneously ovulating, normal, fertile controls. These patterns of endometrial thickness were observed despite significantly higher levels of serum oestradiol-17 beta in all of the hyperstimulated cycles, suggesting that in the normal cycle a maximum response in terms of endometrial development may be achieved. In the three conception cycles endometrial thickness continued to increase throughout the luteal phase, whilst in non-conception cycles plateauing of thickness increase occurred in the mid-luteal phase and reduction in late luteal phase. Whether ultrasonographic evaluation of endometrium during IVF stimulation cycles has any prognostic value regarding prediction of conception has yet to be determined.