Placental protein 12 (PP 12), a decidual protein,in pregnancy complicated by diabetes with retinopathy

Summary Placental protein 12 (IGF-bp/PP 12) is a soluble tissue antigen produced in the decidua. PP 12 was measured in the 3rd trimester of diabetic pregnancy complicated by retinopathy. The mean values of the healthy control group between 28 and 34 and between 35 and 39 weeks gestation were 108±39 g/l and 124±47 g/l, respectively. The equivalent values in diabetic pregnancies complicated by retinopathy were 200±80 g/l and 204 ±81 g/l, respectively; both these PP 12 values were significantly (P<0.05) above the values in the control group. There was no significant difference between benign and proliferative retinopathy. The increased PP 12 levels in the presence of diabetic retinopathy are probably caused by decidual degeneration.     

Serum levels of zinc and copper in hydatidiform mole

Objective The aim of this study was to identify any association between serum levels of trace elements zinc and copper and development of hydatidiform mole.Study design Blood samples of 35 patients with complete hydatidiform mole and 34 pregnant women controls were evaluated for serum levels of zinc and copper. Students t-test was used for comparison of mean values of serum zinc and copper and for demographic variables. p0.05 was considered statistically significant.Results Levels of zinc in serum were found to be significantly higher in hydatidiform mole patients than controls (86.3 vs. 68.7 g/l, p=0.027). Serum copper levels were significantly lower in hydatidiform mole patients than controls (123.8 vs. 147.8 g/l, p=0.039; Table 2).Conclusion This is the first study in the English scientific literature reporting higher serum levels of zinc and lower serum levels of copper associated with complete hydatidiform mole.     

Physiological Induction of the Acrosome Reaction in Human Sperm: Validation of a Microassay Using Minimal Volumes of Solubilized, Homologous Zona Pellucida

Purpose: To develop a method that could accommodatemicrovolumes of solubilized human zona pellucida (ZP) andsperm for assessing the induction of the acrosome reaction. Methods: A microassay using 1 l of 2.5, 1.25, 0.6, 0.3,and 0.125 ZP/l incubated with 1 l of a highly motilesperm suspension for 60 min. As a control and parallelto the microassay a standard acrosome reaction techniquewas performed. Results: No significant differences were observed betweenthe percentage acrosome reacted sperm reported by the twoassays under basal conditions (spontaneous) or after inductionwith a Ca²⁺ ionophore or solubilized ZP. At a ZPconcentration of 0.6 ZP/l, the percentages of acrosome-reactedspermatozoa in both techniques were significantlyhigher compared to the spontaneous acrosome reactionresults, namely, 18% and 17%, compared to 10% and 10%,respectively. An approximately 30% level of acrosomal exocytosiswas induced with 2.5 ZP/l in both methods. Conclusions: This newly devised microtechnique is easy andrapid to perform, is repeatable and facilitates the use ofminimal volumes of solubilized human ZP (even a single ZP)for assessment of the inducibility of the acrosome reaction ofa homologous sperm population.     

Performing ICSI using an injection pipette with the smallest possible inner diameter and a long taper increases normal fertilization rate, decreases incidence of degeneration and tripronuclear zygotes, and enhances embryo development

Purpose: To compare the efficacy of two types of injection pipette used for ICSI, one with a larger (5–7 m) inner diameter and a shorter taper with that inner diameter, and another with the smallest (3–5 m) possible inner diameter and a longer taper with that inner diameter. Methods: Retrieved oocytes at metaphase II stage were injected using one of two types of injection pipette, in 33 and 94 cycles, respectively, in a total of 127 cycles in 108 patients. Results: In comparison to the injection pipette with a larger (5–7 m) inner diameter and a shorter taper with that inner diameter, the injection pipette with the smallest (3–5 m) possible inner diameter and a longer taper with that inner diameter increased normal fertilization rate ((70 ± 3.6)% vs. (86 ± 2.2)%; P = .001; mean ± SEM); decreased the incidence of degeneration ((14 ± 2.4)% vs. (5 ± 1.4)%; P = .001) and tripronuclear zygotes ((1.0 ± 0.35)% vs. (0.1 ± 0.21)%; P = .03); increased Day-2 diploid embryos ((69 ± 3.7)% vs. (85 ± 2.2)%; P = .001) and good-quality Day-2 diploid embryos ((67 ± 4.0)% vs. (79 ± 2.4)%; P = .03), all per injected oocyte; and increased the number of blastomeres per good-quality Day-2 diploid embryo ((3.0 ± 0.21 vs. 3.8 ± 0.12; P = .0003). Conclusions: Performing ICSI using an injection pipette with the smallest (3–5 m) possible inner diameter and a longer taper with thatn inner diameter maximizes normal fertilization rate, minimizes the incidence of postinjection degeneration and tripronuclear zygotes, and enhances embryo development.     

Physiology: Physiological Induction of the Acrosome Reaction in Human Sperm: Validation of a Microassay Using Minimal Volumes of Solubilized, Homologous Zona Pellucida

Purpose: The objective was to develop a method that couldaccommodate microvolumes of solubilized human (ZP) andsperm for assessing the induction of the acrosome reaction. Methods: A microassay using 1 l of 2.5, 1.25, 0.6, 0.3,and 0.125 ZP/l incubated with 1 l of a highly motilesperm suspension for 60 min. As a control and parallelto the microassay a standard acrosome reaction techniquewas performed. Results: No significant differences were observed betweenthe percentage acrosome-reacted sperm reported by the twoassays under basal conditions (spontaneous) or after inductionwith a Ca2⁺ ionophore or solubilized ZP. At a ZPconcentration of 0.6 ZP/l, the percentages ofacrosome-reacted spermatozoa in both techniques were significantlyhigher compared to the spontaneous acrosome reactionresults, namely 18% and 17%, compared to 10% and 10%,respectively. Approximately a 30% level of acrosomal exocytosis was induced with 2.5 ZP/l in both methods. Conclusions: This newly devised microtechnique is easy andrapid to perform, is repeatable, and facilitates the use ofminimal volumes of solubilized human ZP (even a single ZP)for assessment of the inducibility of the acrosome reaction ofa homologous sperm population.     

There is a cutoff limit in diameter between a blastomere and a small anucleate fragment

Purpose : To document the DNA content of blastomeres/fragments from early human preembryos and to determine if there is a cutoff diameter at which a cell should be consi- dered an anucleate fragment rather than a blastomere. Methods : Surplus embryos from in vitro fertilization were used. Individual cells were measured, fixated, and stained for DNA. Results : In day 2 preembryos, only 2% of cells with a diameter <45 m contained DNA, compared with 67% of those 45 m. In day 3 preembryos, 3% of cells <40 m contained DNA, compared with 66% of those 40 m. Conclusions : It is suggested that cells <45 m in day 2 preembryos, and <40 m in day 3 preembryos should be classified as fragments, and cells larger than this, as blastomeres. This may influence the embryo scoring system for in vitro fertilization. We therefore recommend that cells within this critical range should be measured when scoring preembryos for embryo transfer.     

Insulin-like growth factor-binding protein PP12 in ovarian cyst fluid

Summary Elevated levels of insulin-like growth factor-binding protein (IGF-bp) were recently found in the serum of patients with ovarian tumors. We studied the concentration of IGF-bp in cyst fluids of 37 women operated on for benign or malignant ovarian cysts. The levels were elevated (>47 g/l) in 6 of 28 benign, but in none of the nine malignant ovarian cysts. The highest IGF-bp concentration (447 g/l) was observed in a benign serous cystadenoma.     

Routine laser assisted hatching results in significantly increased clincal pregnancies

Purpose: To elucidate the appropriateness of current indications for assisted hatching (AH) in cleavage stage human embryos and to confirm our preliminary findings that only young patients (about 67%) benefit from AH. Methods: Prior to transfer, 2 of 3 embryos selected for ET were subjected to laser assisted hatching (LAH). Control group consisted of patients matched by similar characteristics and protocol except LAH was not performed. Results: The clinical pregnancy rate in women 36 years was 64.9% (24/37) for embryos subjected to LAH but was significantly lower (p = 0.029) in the control (33.3%; 10/30). The implantation rate in women 36 years in the test group was 38.1% (40/105) that was significantly higher than that of the control group (17.5%, 14/80; p = 0.0039). Conclusions: LAH is beneficial for women 36 years but not for women 37 years, for embryos with thin zonae (16) but not with thick zonae (17), and for those with repeated failures (37–50%).     

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.     

The influence of transient hyperprolactinemia on hormonal parameters,oocyte recovery,and fertilization rates in in vitro fertilization

A detrimental effect of transient elevation of plasma prolactin (PRL) during in vitro fertilization (IVF) has not been proven; however, treatment with a dopamine agonist has been suggested. The present study was undertaken to determine if transient, midcycle hyperprolactinemia exerted a deleterious effect on the number of oocytes retrieved or on fertilization of oocytes in vitro. Fifty-three infertile patients with midcycle hyperprolactinemia (PRL>20 g/liter) during ovarian hyperstimulation for IVF were compared with 53 matched controls who remained normoprolactinemic. Mean (±SE) serum PRL levels on the day after hCG were significantly higher in the study group (29.5±1 g/liter) than in the control (13.1±0.5 g/liter) (P<0.0005), whereas the mean estradiol (E₂) concentrations on the same day were not significanily different (4822±287 and 4492±269 pmol/liters, respectively). Fertilization rates (72±4 and 70±4%, respectively) and the mean number of oocytes recovered (4.2±0.3 and 3.7±0.3, respectively) did not differ between the two groups. No correlation was observed between serum PRL and E₂ levels, fertilization rates, or the number of oocytes retrieved in either group. Eleven patients with elevated PRL levels as a result of ovarian hyperstimulation were treated with 2.5 mg bromocriptine daily during the next IVF cycle. Serum PRL levels were significantly lower in the treated (5.6±1.8 g/liter) than in the untreated cycles (35.6±3.1 g/liter) (P<0.0005), whereas serum E₂ concentrations did not differ. Although the mean number of oocytes recovered was significantly higher in the treated (6.2±1.1) than in the untreated (4.7±0.7) (P<0.02) cycles, the fertilization rates were significantly lower when the patients were treated with bromocriptine compared with the previous untreated cycle (55±8.0 and 76.5±7.0%, respectively;P<0.05). Our data demonstrate that a transient elevation of PRL during ovarian stimulation for IVF does not adversely affect the endocrine response, number of oocytes retrieved, or fertilization rates. No improvement in these parameters was observed in bromocriptine-treated cycles. These results do not support the treatment of transient hyperprolactinemia with dopamine agonists in IVF patients.     

Effectiveness of Low Dose of Gonadotropin Releasing Hormone Agonist on Hormonal Flare-Up

Purpose: The hormonal response (flare-up) followingadministration of a standard dose (100 g) or a low dose(25 g) of gonadotropin releasing hormone agonist(GnRH-a) (Triptorelin) was compared in patients prior to an in vitrofertilization (IVF) cycle and during the early follicular phaseof a short-term IVF protocol. Methods: The gonadotroph (FSH, LH) and steroid [estradiol(E₂) and progesterone (P)] flare-up was studied on twoconsecutive cycles in 30 normo-ovulatory women. Patientswere randomized to receive either 25 or 100 g of triptorelinfor three days at the beginning of the first cycle. Then doseswere switched according to a crossing over design in thesecond cycle. Results: No significant difference in the magnitude of FSHand E₂ release could be observed following administrationof the two doses of agonist whereas maximal plasma LHlevel was significantly reduced after injection of 25 g oftriptorelin. Conclusions: As compared to a standard dose, using a lowdose of GnRH-a induces an hormonal flare-up which seemsadequate for an optimal follicular recruitment.     

Zona Thinning with Noncontact Diode Laser in Patients Aged ≤37 Years with No Previous Failure of Implantation: A Prospective Randomized Study

Purpose: Zona thinning (ZT) is a technique used to improve pregnancy rates among patients 38 years old and/or patients presenting previous implantation failure. The objective of the study was to determine whether ZT has a beneficial effect on patients younger than 37 years who are undergoing the first ICSI attempt. Methods: A total of 103 patients submitted to ICSI for the first time and those aged 37 years were divided in a prospective and randomized manner into two groups: group I, patients submitted to ZT (n = 51) (a laser diode with 1.48-m wavelength (Fertilaser) was used for the procedure); group II, patients with no ZT (n = 52). In both groups, embryo transfer was performed on the second day. Results: The age of group I patients (31.8 ± 3.6) did not differ (P = 0.53) from that of group II patients (31.4 ± 3.6). The number of metaphase II oocytes was similar(P = 0.76) for the two groups (group I = 9.12 ± 5.27; group II = 8.67 ± 5.02). The average number of embryos available per transfer of group I (6.14 ± 4.02) did not differ (P = 0.69) from that of group II (5.75 ± 3.83). The number of embryos transferred was similar (P = 0.61) for the two groups (group I = 2.76 ± 0.9; group II = 2.87 ± 0.79). The thickness of the zona pellucida of group I embryos (16.6 ± 2.2 m) did not differ (P = 0.08) from that of group II embryos (17.1 ± 1.7 m). The rate of embryo implantation (20.8%) and the rate of clinical pregnancy per embryo transfer (40.3%) were higher for group II than for group I (17.7% and 33.3%, respectively), but the difference was not significant (P = 0.55 and P = 0.54). Conclusions: These results suggest that ZT in the population aged 37 years and with no previous failure of implantation may have no impact on intracytoplasmic sperm injection success rates.     

The association between chlamydial-specific IgG and IgA antibodies and pregnancy outcome in an in vitro fertilization program

Chammydial-specfic IgG and IgA antibodies were determined by a single serovar (L2) immunoperoxidase assay (IPA) in the serum of all patients that have conceived in an in vitro fertilization and embryo transfer (IVF & ET) progrom (n=106) and in a group of patients that went through the program at the same period of time and did not conceive (n=94). The prevalence rate of elevated IPA IgG (titers1128) and IPA IgA (titers116) specific to chlamydiae was significantly higher (P<0.001) in the IVF&ET pregnancy loss and nonconception groups (failures) versus the IVF&ET term pregnancy group (successes) (74 vs 47%, odds ratio=4.1, and 34 vs 14%, odds ratio=4.3, respectively). Stepwise discriminant analysis revealed that elevated specific chlamydial IgG had the greatest effect on the variance between successes and failures in this study group. Our study indicates the possible role of past or chronic active chlamydiae infection on the take-home baby rate in an IVF&ET program.     

Characterization and prediction of IVF cycles generating “slow-cleaving” embryos

Purpose To characterize and predict cycles generating slowcleaving embryos in in vitro fertilization, 86 cycles were retrospectively divided into two groups (slow, n=41, and fast, n=45 according to whether the number of blastomeres per embryo on day 3 was or > than the mean of the distribution, respectively.Results Cycles generating slowcleaving embryos were treated with luteinizing hormonereleasing hormone agonist before ovarian stimulation for a shorter period (12.1±0.5 versus 15.6±1.1 days; P0.01) and had higher immaturity grade of oocyte-corona-cumulus complexes which resulted in embryos (1.6±0.1 vs 1.3±0.1; P0.05) when compared to cycles producing fastcleaving embryos. Both variables entered in a logistic regression model applied in order to predict the probability of a cycle generating slowcleaving embryos (goodness-of-fit chisquare=180.0, degrees of freedom (df)=80, P=0.4786. This model predicted correctly 86.7% (13 of 15) of cycles generating slowcleaving embryos and 83.3% (10 of 12) of cycles producing fastcleaving embryos when the estimated probability of a cycle producing slowcleaving embryos was 0.7 or 0.3, respectively.Conclusion Shorter treatment with hormone-releasing hormone agonist before ovarian stimulation and higher immaturity grade of oocyte-corona-cumulus complexes which result in embryos are predictive characteristics of in vitro fertilization cycles generating slow-cleaving embryos.     

High-dose misoprostol used in outpatient management of first trimester spontaneous abortion

Aim The aim of this study was to evaluate the efficacy and tolerance of a high dose of vaginal misoprostol for outpatient medical management of missed abortion.Methods Three doses of 400 g misoprostol were administered intravaginally every 4 h daily, for a maximum period of 3 days, to 108 women with uneventful first trimester pregnancy failure.Results A total of 98 women (90.7%) were managed successfully, with 74 (68.5%) of them within the first 24 h. The mean dose of misoprostol administered was 1,113.0 g (range 400–3,600 g), and the mean time required was 19.5 h (range 7–65 h). Only 6 out of 108 women (9.3%) required surgical intervention as the result of retained products of conception. The side effect profile was minimal.Conclusion This protocol of 400 g intravaginally misoprostol every 4 h as three daily doses for a maximum of 3 days, may offer an efficacious and safe alternative to the outpatient management of first-trimester missed abortion.     

First trimester diagnosis of partial mole

Background Partial mole is one of the two distinctive subtypes of hydatidiform mole. It is usually paternally derived triploid conceptions in which embryonal development occurs in association with trophoblastic hyperplasia. The definite diagnosis is confirmed by pathological and cytogenetic studies. Ultrasound might be helpful to diagnose partial mole in the first trimester.Case A 25-year-old woman, gravida 2, para 0-0-1-0, was initially seen for antenatal care at 6 weeks pregnant. Ultrasound was undertaken at 13 weeks pregnancy due to her first fetal anomaly, which demonstrated partial mole and embryonic death. The serum hCG was 190,900 mIU/ml. Suction curettage was performed without complication. Histopathological study confirmed partial mole and cytogenetic study of the placenta revealed an uncommon karyotype, mosaicism of triploid (69,XXX/69,XXY). Serum hCG was declined and negative at 8 weeks. The patient was well and serum hCG remained normal throughout 6 months of follow-up.Conclusion Although the majority of partial mole pregnancies cannot be detected by routine first trimester ultrasound examination, first trimester ultrasound can be helpful in some cases, such as this one. If partial mole is sonographically suspected, it should be confirmed with histopathology and cytogenetic studies. The management is similar to complete mole including prompt evacuation and careful monitoring of hCG.     

Complete hydatidiform mole with coexistent live fetus in dichorionic twin gestation

Case report A 28-year-old patient gravida 3, para 2 was admitted at 16 weeks gestation with the diagnosis of complete hydatidiform mole with coexistent live fetus (CHMCF) in dichorionic twin gestation. The HCG level was 530,000 mIU/ml. The pregnancy was terminated by hysterotomy. Outcome Histopathologic analysis revealed a hydatidiform mole and a 17-week-old male fetus attached to the normal looking placenta by a double-vesseled cord. A final karyotype on cord blood samples confirmed normal 46 XY. Weekly performed serial HCG values showed declining trend and were undetectable by 10 weeks post delivery.     

Serum Progesterone in Predicting Pregnancy Outcome After Assisted Reproductive Technology

Purpose: Our purpose was to determine whether serum progesterone predicts pregnancy outcome after superovulation. Methods: One hundred twenty-three consecutively pregnant patients were divided into three groups: group I, 55 patients following superovulation for assisted reproductive technologies; group II, 23 patients after correction of oligoovulation; and group III, 45 patients who conceived spontaneously. When -human chorionic gonadotropin was positive, progesterone was measured on the same serum sample. A serum progesterone level of 45 m/L was set to differentiate between nonviable pregnancy and ongoing pregnancy. Results: In group I, zero (0%) of 38 ongoing pregnancies and 10 (59%) of 17 nonviable pregnancies were observed with a progesterone level of <45 m/L [14.2 ng/ml (P < 0.001)]. In group II, 4 (27%) of 15 ongoing pregnancies and 5 (63%) of 8 nonviable pregnancies had a progesterone level of <45 m/L (P = NS). In group III, 10 (42%) of 24 ongoing pregnancies and 15 (71%) of 21 nonviable pregnancies were observed with a progesterone level of <45 m/L (14.2 ng/ml) (P = NS). Conclusions: A serum progesterone level of <45 nM predicts nonviable pregnancy after superovulation for assisted reproductive technology.     

Correlation of follicular diameter with oocyte recovery and maturity at the time of transvaginal follicular aspiration

Forty-four consecutive patients undergoing transvaginal follicular aspiration for in vitro fertilization underwent ultrasonic measurement of follicular diameter at the time of oocyte retrieval to determine the correlation of follicular size with recovery rates and oocyte maturity. Based on the results of 412 follicles aspirated, the data were grouped by size (11, 12–14, 15–17, 18–20, and 21 mm) and oocyte maturity. Recovery rates were significantly higher in 18- to 20-mm follicles (P<0.01) and lower in those 11 mm (P<0.001). The probability of retrieving a metaphase I or II oocyte was significantly lower in follicles 11 mm (P<0.001), somewhat higher in 12- to 14-mm follicles (P<0.01), and equally high among the other groups. There were no differences in the incidence of fractured zonas. We conclude that follicles 15 mm provide the highest probability of retrieving mature oocytes and the low recovery rates of mature oocytes from follicles 11 mm suggest that, in selected circumstances, the operating surgeon may choose not to aspirate them.     

Development of murine embryos following electroporation

Purpose: The objective was to establish the parameters for reversible electroporation of murine embryos. Methods: In Trial 1, murine presumptive zygotes received an electrical pulse of 5, 10, or 20-s duration, and one of five voltages (100, 200, 250, 300, or 400 V). In Trial 2, embryo orientation within the electroporation chamber was evaluated with 250 or 400 V at a pulse period of 10 s. Results: Presumptive zygotes that received 400 V at each pulse length and zygotes exposed to 20 s at each voltage had the lowest embryonic development (P < 0.05). Presumptive zygotes that received 250 V had higher development compared to 400 V, irrespective of orientation (P < 0.01), but development was lower than the controls (P < 0.01). Conclusions: Electrical stimulation of presumptive zygotes can have a detrimental impact on early embryo development, but low amounts of stimulation may allow for potential gene transfer in transgenic experimentation.