The matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade protein components of the extra-cellular matrix. The necessity of breakdown of physical barriers in the fertilization process suggests that MMPs, along with their tissue inhibitors (TIMPs), might be involved in this task. We have examined the presence of MMP and TIMP in normal and abnormal human sperm samples by gel zymography and Western blot analysis. Thirty-five normal sperm samples and 35 abnormal sperm samples were examined in this study. Gel zymography showed 92-, 72-, 62-, and 28-kd molecular-weight bands exhibiting gelatin-degrading activity in both normal and abnormal sperm samples. The 92-, 72-, and 62-kd bands with gelatinolytic activity are consistent with pro-MMP-9, pro-MMP-2, and active MMP-2, respectively (pro-MMP being the zymogen of MMP). Western blot analysis showed the presence of TIMP-1 in both normal and abnormal sperm samples. A higher 28-kd activity and a lower 92-kd MMP activity in normal sperm samples relative to abnormal samples were detected. No marked difference in TIMP-1, 72-kd, and 62-kd release was observed between normal and abnormal sperm samples. In conclusion, this is the first report of MMP activity in normal and abnormal human sperm samples and of TIMP presence in sperm samples. The data indicate a different MMP profile between normal and abnormal sperm samples, with a higher 28-kd activity and a lower 92-kd MMP activity in normal relative to abnormal samples.

Mitogen-activated protein (MAP) kinase in human eggs has been investigated by using immunoblotting with both anti-Active MAPK and anti-ERK2 antibodies. The results showed that the main form of MAP kinase was p42ERK2. It was in a dephosphorylated form in oocytes at the germinal vesicle stage, but fully phosphorylated in unfertilised mature eggs. MAP kinase phosphorylation was significantly decreased when pronuclei were formed after intracytoplasmic sperm injection. Neither MAP kinase expression nor activity was detected in morphologically degenerated eggs. Although MAP kinase still existed in early embryos arrested at the 8-cell or morula stages, little, if any, activity could be detected. These data suggest that MAP kinase may play an important role in the cell cycle regulation of human eggs, as in other mammalian species.

The necessity of epidermal growth factor (EGF) in the process of mouse embryo development and outgrowth in vitro was studied. Mouse 4-cell stage embryos were cultured up to spreading stage (outgrowth) in human tubal fluid (HTF) medium alone (control) or with 10 ng/ml EGF and 1:250 diluted monoclonal antibodies against EGF (study groups). Hatching and outgrowth were significantly increased up to 60.9 and 52.4% respectively, while in the control only 33.7 and 20.4% reached hatching and outgrowth respectively. Moreover monoclonal antibodies against EGF significantly inhibited embryo development (P < 0.01). Only 5.8% of the embryos reached the hatching stage and none of them reached the spreading stage. Our results show that EGF is probably involved in the modulation of early embryonic growth and in the initiation of implantation.

Objective: To use injection of spermatids into oocytes as a mode of infertility treatment in cases in which spermatozoa are not available.Design: Prospective clinical evaluation and case reportSetting: In Vitro Fertilization Unit, Herzliya Medical Centers, Herzliya-on-Sea, Israel.Patient(s): Thirteen couples with male factor infertility in which the male partner lacked spermatozoa in the ejaculate or testicular biopsy samples.Intervention(s): Round spermatid injection and elongated spermatid injection into oocytes.Main Outcome Measure(s): Evaluation of the rate of two-pronucleated and single-nucleated zygote development.Result(s): The rate of two-pronucleated zygote development after round spermatid injection and elongated spermatid injection was relatively low (27% and 36%, respectively). Single-nucleated zygotes develop more frequently after round spermatid injection and elongated spermatid injection (35% and 17%, respectively) than after intracytoplasmic sperm injection with mature spermatozoa. A normal pregnancy and childbirth resulted from the transfer of 4 cleaving embryos, each of which developed from a single-nucleated zygote in a round spermatid injection treatment cycle with ejaculated spermatids.Conclusion(s): Embryos derived from single-nucleated zygotes after spermatid conception can be viable and give rise to an ongoing clinical pregnancy and childbirth. (Fertil Steril(R) 1998;70:67-70. (C)1998 by American Society for Reproductive Medicine.).

The study was conducted to examine whether the presence of glucose in the incubation medium affects fertilization, development and implantation rates of human oocytes of patients who were attending our in-vitro fertilization programme. Harvested oocytes were transferred into one of four different media: human tubal fluid (HTF), P1, M3 and IVF-Universal (IVF-Med). Three of these contained glucose; the fourth (P1), contained no glucose or phosphate ions. In an independent preliminary study, some of the oocytes of each patient were incubated in IVF-Med, which lacks phosphate ions, but not glucose. Comparisons of fertilization rates between media pairs showed differences among all pairs except HTF and M3. When comparing the four study groups, no difference was noticed in embryo development or embryo quality 48 h post-ovum retrieval. A higher development rate was demonstrated in embryos incubated in M3 medium, in comparison with the P1 and IVF-Med embryos after incubation for 72 h. No difference in pregnancy rate was found after embryo transfers of preimplantation embryos which were incubated in one of the following media: HTF, M3 and IVF-Med (seven out of 22, 18 of 54 and 32 of 69 treatment cycles respectively). A lower incidence of pregnancies occurred following transfers of embryos which were incubated in P1 medium (seven pregnancies out of 37 cycles). We suggest that the presence of glucose in the incubation medium enhances implantation potential of in-vitro-developing preimplantation embryos.

OBJECTIVE:To investigate the use of a simplified short-term coculture system with luteinized granulosa cells (GCs) in patients with failed IVF-ET.DESIGN:Controlled clinical study.SETTING:IVF unit, Department of Obstetrics and Gynecology, Carmel Medical Center.PATIENT(S):Patients with poor embryo quality in their previous IVF-ET cycles.INTERVENTION(S):Embryos from 40 patients, in which > 50% of the embryos were classified as poor quality in their previous IVF attempts, were grown on autologue GC culture system for a short period (24-48 hours) before being replaced in the uterine cavity.MAIN OUTCOME MEASURE(S):Embryo quality.RESULT(S):Significant decrease in poor quality embryos and increase in the proportion of good quality embryos were observed using a coculture system with autologue human GCs. Pregnancy rates in this groups of patients reached our standard IVF results during the same period.CONCLUSION(S):This study describes a simplified short-term coculture system with human autologue GCs. Poor quality embryos may be rescued to cleave regularly.

In a previous study (Svirski et al., 1993), it was found that growth inhibition ofGracilaria spp., when cultured in the presence ofUlva cf.lactuca, was not due to shading or nutrient depletion, but seemed to be caused by competition for inorganic carbon or some type of allelopathy. In the present study, we attempted to differentiate between these two possible influences by (1) growing the two algae in biculture under various conditions, but keeping inorganic carbon levels constant and measuring net photosynthesis, respiration and growth rates, and by (2) measuring growth rates ofGracilaria spp. in the presence of extracts derived from media previously used to growUlva cf.lactuca.

ABSTRACT A theoretical framework on the combined effect of water velocity and solute concentration on the photosynthetic performance of the red alga Gracilaria conferta ( Rhodophyta) is developed. This is based on the balance between the rate of transport through boundary layers and Michaelis‐Menten‐type equations for carbon consumption and for production of oxygen and hydroxyl ions. By comparing the theoretical models with experimental data, we found that the mechanism of enhancing photosynthetic rates by increasing water velocity cannot be attributed to enhanced bicarbonate and CO 2 transport, nor to CO 2 as a sole source of carbon. Velocity‐facilitated photosynthesis may be due to the enhanced removal of OH − ions, which inhibit photosynthesis when accumulated on the algal surface. Oxygen had no inhibitory effect on Gracilaria conferta.

To investigate the effect of co-treatment with growth hormone (GH) for ovulation induction with human menopausal gonadotrophins (HMG) on conception, we compared the pregnancy rate and response to co-treatment with GH versus HMG/human chorionic gonadotrophin (HCG) alone in a prospective, randomized, cross-over protocol of ovulation induction for either in-vivo or in-vitro fertilization (IVF). The main outcome measures were the amount of gonadotrophin used and conception. Co-treatment with GH was associated with a reduction of approximately 30% in gonadotrophin requirement. In 24 clonidine negative patients 14 pregnancies were achieved (58.3%) either in the GH/HMG/HCG cycle or in the succeeding one. GH co-treatment did not generate any pregnancy in eight clonidine positive patients. We conclude that growth hormone may increase the pregnancy rate when combined with HMG/HCG for ovulation induction, not only in the co-treatment cycle but also in the succeeding one. The beneficial, synergistic effect of GH co-treatment was detected in clonidine negative but not in clonidine positive infertile patients.

The present study was undertaken to assess whether the increase in serum progesterone concentration following the administration of human chorionic gonadotrophin (HCG) may have predictive value on the in-vitro fertilization (IVF) success rate. Progesterone concentration on the day of HCG administration and the increase in progesterone concentration on the following day were evaluated in 140 consecutive patients undergoing IVF with embryo transfer. Stimulation protocol in all study patients entailed intranasal administration of short-acting gonadotrophin-releasing hormone agonist (GnRHa) buserelin and human menopausal gonadotrophin. A pregnancy rate of 37.2% was achieved when at least three embryos were transferred. The only significant difference between conception and non-conception cycles was found in serum progesterone concentrations after HCG administration (P < 0.01), whereas the mean progesterone concentration on the day of HCG did not differ. No difference in other hormonal or cycle parameters was observed. The increase in progesterone concentration was significantly greater in the group of patients who achieved pregnancy than in the group who did not (2.2 +/- 0.2 versus 1.6 +/- 0.1 ng/ml, respectively; P < 0.01). A critical breakpoint in serum progesterone was arbitrarily determined at 1 ng/ml. An increase in progesterone concentration > or = 1 ng/ml when three or more embryos were transferred was associated with a positive predictive value for pregnancy of 40.4% (sensitivity of 94.7%), whereas a negative predictive value of 86.7% was obtained when this value was < 1 ng/ml. These findings indicate that an adequate rise in serum progesterone following HCG administration provides useful information about the possible outcome of the treated cycle.

Objective: To investigate the morphology and proliferative ability of cumulus and granulosa cells (GCs) originating from cystic follicles and normal-sized follicles after ovarian stimulation.Design: Granulosa cells, cumulus cells, and follicular fluid (FF) were aspirated from cystic follicles and normal-sized follicles from the same ovary. Morphology and proliferative ability of cumulus and GCs were assessed by Giemsa stain and thimidine incorporation, respectively. Cell proliferation was assessed in medium or FF originating from cystic follicles or normal-sized follicles.Results: An oocyte was found in 40% of the cystic follicles versus 68% in the normal-sized follicles. Changes in dispersion and adhesion properties were observed in cystic versus normal aspirated corona cumuli complex. Proliferative ability was consistently lower in GCs originating from cystic follicles versus normal-sized follicles. Proliferation of GCs originating from normal-sized follicles or cystic follicles was inhibited or increased when grown in FF from cystic follicles or FF from normal-sized follicles, respectively. Differences in embryo quality were significantly in favor of oocytes originating from normal-sized follicles. Although the fertilization rate of those oocytes appeared to be higher, the difference was not of statistical significance.Conclusions: Inhibition of GC proliferation in FF from cystic follicles can be reversed by incubating cells in FF from normal-sized follicles. We conclude that factors in the FF may affect cell proliferation.

Objective: To investigate P secretion by granulosa cells (GCs) versus cumulus cells derived from human preovulatory follicles.Design: Cells were recovered by aspiration of preovulatory follicles in 44 women participating in an IVF program. Induction of ovulation was performed using clomiphene citrate, hMG and hCG (group I), hMG/hCG (group II), buserelin acetate/hMG/hCG (group III), or Decapeptyl/hMG/hCG (group IV).Setting: Laboratories of the IVF Unit at the Department of Obstetrics and Gynecology, Carmel Medical Center, Haifa, Israel.Main Outcome Measures: Secretion of P was examined after cultures for 96 hours under nonstimulated and hCG stimulated conditions.Results: Progesterone secretion by GCs derived from all four groups was found to be higher compared with the respective cumulus cells. However, although the ratios of P secretion by GCs versus cumulus cells in groups I, II, and III were very similar, a significantly lower value was observed in group IV. The response of GCs to hCG in terms of P secretion was higher with at least one dose of hCG in groups I and IV compared with groups II and III. The response of cumulus cells to hCG was absent regardless of the treatment protocol used in vivo.Conclusion: Our results demonstrate that in the human preovulatory follicle, GCs and cumulus cells differ in their capacity to secrete P as well as in their response to hCG. They further suggest that the mode of induction of ovulation affects the relative capacity of GCs and cumulus cells to secrete P and their ability to respond to hCG.

Bulk water velocities and local relative velocities generated in experimental tanks around and within thalli of free moving Gracilaria conferta were estimated according to the dissolution rate of benzoic acid sticks. Boundary-layer thickness and HCO 3 − -mass-transfer coefficient were derived from the water velocities. Average relative velocities varied between 12 cm s −1 to less than 0.1 cm s −1 as a function of the absolute water flow in the tank, alga shape and location within the thallus. The lower range of velocities was observed at 20% of maximum aeration in the inner part of the plant. In laboratory experiments, photosynthetic rates, as determined in a closed Clark-type O2-electrode system, increased by 30%–50% when water velocity was increased from zero to about 1.5 cm s −1. Another minor increase was obtained between 1.5 cm s −1 and 8 cm s −1 water velocity. This response to water motion was affected by bulk inorganic carbon concentration and by plant condition, as was reflected from the differences in the response in the winter and spring. It might be suggested that under carbon saturation, water velocity above 2 cm s−1 provided almost sufficient flow to saturate carbon uptake.

The effect of epidermal growth factor (EGF) on embryonic growth, development, attachment and spreading in vitro was studied. EGF was added to 130 embryos at the 4-cell stage; to 128 embryos at the blastocyst stage; and to 147 embryos 24 h following spreading. Development of embryos from the 4-cell to the blastocyst stage, differentiation of the inner cell mass (ICM) and trophectoderm, and the occurrence of attachment and spreading were evaluated. Embryo development was significantly inhibited in cultures supplemented with 100 ng/ml EGF compared to the controls (P < 0.001). Development of 4-cell embryos to blastocysts occurred in 25% of the EGF group compared to 85% of controls. Spreading occurred in 20% of 4-cell embryos and 30% of blastocysts treated with EGF, compared to 80 and 90% of corresponding controls. In embryos developing from the 4-cell stage, massive growth of the ICM and inhibition of the trophectoderm occurred, whereas both ICM and trophectoderm were inhibited by EGF in embryos developing from the blastocyst stage. Following spreading, EGF caused massive growth of the ICM and regression of the trophectoderm. Our preliminary results show that EGF may be involved in the modulation and control of early embryonic growth and differentiation.

Gamete intrafallopian transfer involves a direct transfer of both human gametes, sperm and oocytes, into the fallopian tube. Since the first report of a successful pregnancy following the use of this technique by Asch et al in 1984, its role in the treatment of infertile women with patent tubes has been established. Recent data demonstrate a 34.4% clinical pregnancy rate, with the highest pregnancy rate achieved in infertile women due to endometriosis or unexplained infertility.

The effect of follicular aspiration and oocyte retrieval on hormonal parameters was examined in women undergoing ovarian stimulation for in-vitro fertilization (IVF) compared to induced ovulation in women undergoing ovarian stimulation for intrauterine insemination (IUI). Blood samples were collected immediately before and 1 h after oocyte retrieval and 48 h later on the day of embryo transfer in 25 IVF patients and before the insemination and 48 h later in 20 IUI patients. A highly significant fall in serum levels of oestradiol (E2), progesterone (P) and human chorionic gonadotrophin (HCG), (P less than 0.001) was observed in the IVF group 1 h after follicular aspiration. The decline in serum E2 levels was maintained at 48 h. In contrast, there was no significant change in serum E2 levels in the IUI group during 48 h. The immediate decline in E2 levels after follicular aspiration might play a role in preventing ovarian hyperstimulation syndrome.

The present study was undertaken to determine whether the sonographic characteristics of the endometrium in the proliferative phase of the natural cycle combined with meticulous hormonal monitoring would have an impact on the prediction of conception. Fourteen women with regular ovulatory cycles were examined daily, from day 8 of the cycle, by transvaginal ultrasound through 16 unstimulated cycles. All patients had had at least four previous unsuccessful donor artificial insemination cycles scheduled by basal body temperature records. During the study, donor artificial insemination was meticulously timed by hormonal and sonographic monitoring. Four clinical pregnancies occurred with a success rate of 29% per patient, or 25% per cycle. The endometrial thickness at the mid-proliferative phase (day 8) was significantly smaller in patients who conceived in comparison with patients who failed to conceive (0.33 +/- 0.07 cm vs. 0.59 +/- 0.03 cm; p < 0.01). The endometrial thickness on the day of the luteinizing hormone (LH) peak was not significantly different between the two groups. However, the amount of endometrial growth between day 8 of the cycle and the day of the LH peak was significantly greater in conception than non-conception cycles (0.55 +/- 0.05 cm and 0.24 +/- 0.06 cm; p < 0.01). No difference in hormonal parameters was observed either on day 8 of the cycle or on the day of the LH peak between conception and non-conception cycles. On the day of the LH peak, an endometrium with typical triple line appearance and thickness of 0.6 cm or more was seen significantly more often in conception than non-conception cycles (p < 0.05). No pregnancy occurred with an endometrium < 0.6 cm at the time of ovulation. The presence of the favorable endometrial pattern in the periovulatory phase was associated with a positive predictive value for pregnancy of 50% (sensitivity of 100% and specificity of 67%), whereas the negative predictive value of the hyperechoic pattern at the time of ovulation was 100%. Our data show negative correlation between early growth of the endometrium and pregnancy in unstimulated cycles, suggesting that the amount of endometrial growth during the cycle up to ovulation may have an important role in implantation. The sonographic image of the endometrium in natural cycles provides useful information about the possible outcome of the treated cycle.

The performance of two different transfer catheters has been evaluated in 193 consecutive embryo transfers. Forty pregnancies were achieved, corresponding to a pregnancy rate of 21% of all embryo transfers. The Frydman catheter was used in 123 attempts whereas 70 embryo transfers were performed with the Tom Cat catheter. The Tom Cat catheter yielded a significantly higher pregnancy rate than the Frydman catheter (28% versus 16%; P = 0.03). The mean number of embryos transferred per attempt was similar in the two groups (2.9 +/- 0.1 and 3.0 +/- 0.1 in the Tom Cat and Frydman groups respectively) as was the quality. When more than three embryos were transferred, a pregnancy rate of 37% per embryo transfer was achieved using the Tom Cat versus 20% using the Frydman catheters (P = 0.03). A higher frequency of difficult transfer procedures was observed when the Frydman catheter was used while the Tom Cat performed much better in difficult cases. An alternate use of the two catheters suggests a significantly better performance of the Tom Cat catheter in relation to the efficiency of establishing pregnancy.