Factors affecting survival and implantation if cryopreserved human embryos

Five- to ten-cell embryos and expanded blastocysts from 68 patients were thawed in an attempt to establish pregnancy. Three fresh embryos had been replaced unsuccessfully in these patients. Forty-five patients had intact freeze-thawed embryos replaced and 12 became clinically pregnant. Ten of these pregnancies have now advanced beyond week 24. The preliminary results presented here demonstrate that significantly more expanded blastocysts survive cryopreservation than cleaving embryos. Faster-growing embryos did not survive better than slowly growing embryos, but the incidence of implantation was higher with faster-developing embryos. Significantly more blastocysts with a normal morphology survived cryostorage than those scored as irregular (77 versus 7%). A similar trend was observed when normal and irregular cleaving embryos were frozen, but the difference was not significant. The severity of contraction of blastocysts upon the addition of cryoprotectant increased the incidence of survival. The proportion of patients whose embryos had holes in the zona pellucida following cryopreservation was significantly higher when embryos were frozen at the cleaving stage (39%) rather than the expanded blastocyst stage (16%). Almost all embryos with damaged zonae degenerated. The proportion of cleaving embryos that survived cryostorage was inversely correlated with the number of follicles aspirated.     

Effects of charcoal-extracted serum as a growth medium supplement on in vitro development of mouse embryos

Two-cell mouse embryos (N=952) were cultured in modified Ham's F-10 medium supplemented with 15% charcoal-extracted serum or 15% nonextracted serum from 17 patients. Each sample was assayed independently and all experiments run in duplicate. Ten nonextracted samples inhibited development to the blastocyst stage compared to controls in F-10 alone. Charcoal extraction significantly improved (P<0.05) development compared to nonextracted serum in eight of these samples. Seven samples supported development and no difference was noted between F-10 alone and extracted and nonextracted serum-supplemented media in this group. In conclusion, charcoal extraction significantly reversed the embryotoxic effects of some sera in the two-cell mouse embryo model.     

Bovine serum albumin (BSA) can replace patient serum as a protein source in an in vitro fertilization (IVF) program

Alternate protein sources have been suggested to replace the commonly used cord or patient serum for in vitro fertilization (IVF) procedures. During an 11-month period 127 patients treated for in vitro fertilization had elther their serum (N= 71) or bovine serum albumin (BSA;N= 56) used as the protein source in the insemination and growth media. Ham's F-10+0.5% BSA was used for sperm swim-up and insemination media and 1% BSA was used for the growth media. Patient's serum was added to Ham's F-10 culture media at concentrations of 7.5 and 15% for insemination and growth, respectively. Embryo transfer was performed with Ham's F-10 containing 90% maternal serum in both groups. Fertilization rate of 259 oocytes inseminated in medium containing patient's serum did not differ when compared with 200 oocytes inseminated in medium containing BSA. Likewise, rates of obnormal fertilization, cleavage, and pregnancy were similar in both groups. In a second experiment, 148 normally fertilized oocytes were transferred after 24 hr in culture to growth media containing two different concentrations of BSA (0.5 or 1%). Cleavage rates for the two groups were similar and the percentage of embryas developed to 4 cells did not differ signficantly. We conclude that a single concentration of BSA can safely be used to supplement culture media in human IVF with several practical and economical benefits.     

Turner syndrome with pseudodicentric Y chromosome mosaicism

The objective was to compare the impact of gonadal cell line upon the phenotype of a Turner syndrome patient with mosaic karyotypes. A 10-year-old female presented with typical Turner syndrome. Chromosomal analysis of lymphocytes revealed 45,X (16%)/46,X,pseudodicentric Y (p terq12::q12p ter) (84%). Karyotype of the gonads revealed 45,X (85%)/46,X,pseudodicentric Y (p terq12::q12p ter) (15%). Discrepancy of the individual cell lines between the lymphocytes and the tissue might exist. The phenotype of patients with mosaicism is related with the individual proportions of gonadal cell lines.     

Globulins in Protein Supplements Promote the Development of Preimplantation Embryos

Purpose: Our purpose was to investigate the effect of- and-globulins contained in protein supplements on the development of preimplantation embryos. Methods: Mouse one-cell embryos were cultured in medium supplemented with 4 mg/ml human serum albumin (HSA), 4 mg/ml HSA plus human globulins (0.2, 0.4, 0.8, and 1.6mg/ml) that consisted predominantly of- and -globulins, or 10% Plasmanate Cutter (PC). Blastocysts developed in media supplemented with these various protein sources were stained with Hoechst 33342 to determine the number of cells. Results: Supplementation with 0.4 to 1.6 mg/ml globulins or PC significantly increased the rate of blastocyst development compared with that observed with the addition of HSA. Supplementation with globulins significantly increased the hatching rate in a dose-dependent manner. The number of cells in the blastocysts was significantly increased when the embryos were cultured with 0.8 mg/ml of the globulins or PC. Conclusions: The present observations suggest that - and -globulins in protein supplements promote embryo development and hatching.     

Prehysterectomy embolisation of uterine arteries for large uterine fibroids—a report

Hysterectomy for large uterine fibroids can prove to be a technically difficult procedure. The irregular shape of the uterus filling the pelvis may make access to the pedicles difficult. The larger the size of the uterus, the greater the risk of significant blood loss and trauma to the surrounding structures. Uterine artery embolisation (UAE) has been in use since 1991 as a noninvasive treatment for uterine fibroids and has had encouraging results. Various workers have reported success rates between 86% and 100%. We present here our experience with two patients who underwent embolisation of the uterine arteries, followed by hysterectomy. In both cases, patients had symptomatic uterine fibroids and no desire to conceive. The patients were counselled about the combined treatment and other treatment alternatives available to enable them make an informed choice.     

Comparison of the Media for Isolation and Storage of Round Spermatid Nuclei Before Intracytoplasmic Injection

Purpose: This study investigated whether K ⁺ -rich medium is better than pure NaCl solution or Na ⁺ -rich cell culture medium for handling round spermatid nuclei prior to injection into oocytes (ROSNI). Methods: Round spermatids of the mouse were isolated and stored in isotonic NaCl, a cell culture medium (CZB), or a nucleus isolation medium (NIM) before injection into oocytes. The rates of normal fertilization, embryonic development in vitro, and birth of normal offspring after transfer of embryos to foster mothers were determined. Results: In vitro development of ROSNI-produced zygotes to blastocysts was the same when naked spermatid nuclei were exposed briefly to three media. However, a long (60-min) exposure of the nuclei to NA ⁺ -rich medium was detrimental. In K ⁺ -rich NIM naked spermatid nuclei best retained their ability to participate in normal embryonic development. Conclusion: NIM was better than Na ⁺ -rich medium for retaining isolated spermatids competent to participate in normal embryonic development.     

The relation between depth of trophoblastic invasion and β-HCG levels in tubal pregnancies

-HCG (human chorionic gonadotropin) values of over 2500 I.U./l are associated with higher failure rates for therapy with prostaglandin F2 alpha in tubal pregnancies. The purpose of our study was to ascertain if the 2500 I.U./l limit correlates with histopathology. We therefore compared the pre-operative -HCG-values and intraluminal and extraluminal trophoblast growth in tubal pregnancy. Purely intraluminal trophoblast was significantly more frequent in patients of group I (-HCG < 2500 I.U./1), while group II patients (-HCG > 2500 I.U./l.) almost exclusively had extraluminal growth (P=0.0045). Since the efficacy of prostaglandin F2 alpha therapy depends on intact tubal musculature the correlation of the -HCG threshold level with histopathologic findings may explain the high failure rate in patients with -HCG values above 2500 I.U./l. Correspondence to: M. Klein     

Cryopreservation of in vitro-fertilized human embryos: Histologic and cytogenetic analysis of an ectopic conceptus

In this study, 39 embryos from 17 patients were cryopreserved in a Planer R204 cell freezer using the protocol of Mohret al. (J Vitro Fert Embryo Transfer 21-10, 1985). The procedure was modified by supplementing the cryoprotectant with 10% heat-inactivated and filtered (0.22 m) maternal serum instead of fetal calf serum, and embryos were frozen in 500-l plastic straws instead of glass ampoules. After 12–25 weeks of storage in liquid nitrogen, 12 embryos from six patients were thawed at 8.0°C min to room temperature, incubated in 75% maternal serum with Ham's F-10, and replaced in utero. One pregnancy occurred. The patient was a 34-year-old nulligravida with occluded fallopian tubes. A year prior, she conceived triplets from three embryos during an in vitro fertilization (IVF) cycle, but she delivered at 21 weeks and the infants did not survive. The second IVF attempt produced four embryos. Two were replaced during the IVF cycle, but they did not implant. Two were cryopreserved and replaced 25 weeks later. On day 28 after replacement, beta human chorionic gonadotropin (-hCG) was 4126 IU, but there was no gestational sac in utero on ultrasonographic examination. Laparoscopy disclosed a right tubal pregnancy which was removed with the fallopian tube. Histological examination demonstrated normal chorionic villi. The chromosomal pattern was 46 XX by direct analysis and cell culture.     

Adjuvante Chemotherapie beim Mammakarzinom

Zusammenfassung Die Effektivität der adjuvanten Chemotherapie wie der antihormonellen Therapie sind in der letzten Dekade entscheidend verbessert worden. Sequenzielle chemoendokrine Therapien haben zu einer zusätzlichen Wirkungssteigerung bei hormonrezeptorpositiven Patientinnen geführt.Während CMF und anthrazyklinhaltige Schemata schon lange routinemäßig eingesetzt werden, ist das optimale anthrazyklinhaltige Regime durch St. Gallen 2005 erstmals klar benannt worden (6× FEC oder FAC). Nach Datenlage verfügen wir mittlerweile über 4 Wirksamkeitsstufen der adjuvanten Chemotherapie (4× AC oder 6× CMF <6× FEC(FAC) < taxanhaltige Regime < dosisdichte Regime). Dieser Zugewinn durch die Hinzunahme neuer Substanzen oder die Änderung des schedule (dosisdichte Therapie) ist in randomisierten Studien belegt.Kontrovers diskutiert wird, ob es hochhormonrezeptorpositive Patientinnen in der mittleren Risikosituation gibt, bei denen die alleinige antihormonelle Therapie eine Alternative zur sequenziellen chemoendokrinen Therapie darstellen kann. Dies impliziert weitreichende Konsequenzen hinsichtlich der Therapiekosten, der Therapiedauer, dem Nebenwirkungsspektrum und noch mehr als zuvor der Aufklärung und der geeigneten Selektion der Patientinnen.     

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.     

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.     

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.     

Human early pregnancy factor: serum concentrations before and after therapeutic abortion in comparison with β-hCG and an early pregnancy associated protein

Summary Serum concentrations of the early pregnancy factor (EPF), -hCG and the early pregnancy associated protein (EPAP) were measured in 12 patients before and after therapeutic abortion for social-medical reasons. Detection of EPF was performed by the rosette-inhibition assay, -hCG quantification by radioimmunoassay and EPAP measurements by rocket immunoelectrophoresis using a monospecific polyclonal rabbit antiserum. The disappearance rate of EPF after termination of pregnancy was closely correlated with the decrease of -hCG concentrations and loosely correlated with the decrease of EPAP concentrations . No correlation has been found between hCG and EPAP values.     

Prostaglandin metabolism in umbilical vessels of smoking and non-smoking mothers

Summary Specimens of saliva were obtained from 106 women with normal pregnancies and from 42 women, whose children turned out to small for dates (birthweight below the 10th centile). Saliva estriol was highly significantly (P<0.0001) decrease in the group with a newborn with a birthweight below the 10th centile (500340 ng/ml, meanSD) when compared with the group with infants whose birthweight was over the 10th centile (813449 ng/ml). Estriol determination in saliva seems to be a simple, non invasive method of aiding the detection of fetal growth retardation.     

Identification of biologically active inhibin in the peritoneal fluid of women

Purpose Immunoreactive inhibin (i-inhibin) has been reported to be present in the peritoneal fluid of women. The radioimmunoassay employed measures free, biologically inactive -subunits(s) equally as well as dimeric, biologically active inhibin. The present study was designed to determine if biologically active, dimeric inhibin is present in the peritoneal fluid of women.Methods Peritoneal fluid of four women was assayed by radioimmunoassay, a sheep pituitary bioassay, and two ELISA procedures which utilized specific monoclonal antibodies for the capture of the -subunit (ELISA-A) or the -subunit (ELISA-B) of inhibin and subsequent quantification of dimeric inhibin-A.Results There was a good correlation between the values obtained by radioimmunoassay, bioassay, and both ELISAs; two samples (from the late follicular phase) with relatively high i-inhibin concentrations were positive in all four assays, whereas two samples (from the early follicular phase) with very low i-inhibin concentrations were negative in the bioassay and ELISAs.Conclusion A significant portion of the immunoreactive inhibin in the peritoneal fluid obtained during the late follicular phase of women is dimeric, biologically active inhibin. We speculate that this may have potential implications for oocyte maturation and early embryogenesis within the oviduct.Presented at the 50th Annual Meeting of the American Fertility Society, San Antonio, Texas, November 5–10, 1994.     

Platelet specific protein (β-thromboglobulin and platelet factor 4) in normal pregnancy and in pregnancy complicated by preeclampsia

Summary Pregnancy is accompanied by significant alterations of platelet function. Platelet activity can be determined by measurement of plasma levels of secreted platelet proteins. In this study we determined plasma levels of -Thromboglobulin (-TG) and Platelet Factor 4 (PF4) simultaneously in 35 women with uncomplicated pregnancy and in 15 patients with preeclampsia in third trimester of gestation. Additionally, PF4 plasma levels were measured using a commercially available Radio Immunoassay (RIA) and an Enzyme Immunoassay (EIA) simultaneously and values obtained were compared. Platelet count and creatinine were in the normal range in both groups; however, significantly higher levels of -TG (P<0.0005) and PF4 (P<0.0001) were found in case of preeclampsia. High levels of platelet proteins emphazise the active role of the platelets in the alterations of hemostasis in cases of preeclampsia.     

Beware: pregnancy and second-generation endometrial ablation

With the promotion of second-generation endometrial ablative techniques—namely thermal balloon endometrial ablation and microwave endometrial ablation—as alternatives to hysterectomy for dysfunctional uterine bleeding, with phrases like three-minute hysterectomy, some women become complacent about contraception after ablation. Some even assume that hysterectomy, as widely used in the popular press, means no more children. Pregnancy after endometrial ablation does not represent a failure of the technique. The failure is due to poor counselling or to women not taking the advice to use appropriate contraception after the procedure.     

Modifying effects of epidural analgesia or general anesthesia on the stress hormone response to laparoscopy for in vitro fertilization

Modifying effects of epidural analgesia and general anesthesia on stress hormone release was studied during laparoscopy for in vitro fertilization (IVF). In 24 women follicle development was stimulated by clomiphene and gonadotropin treatment, and oocytes were collected by laparoscopy under epidural analgesia in 11 women and under fentanyl-supplemented nitrous oxide-oxygen anesthesia in 13. The plasma levels of immunoreactive -endorphin (ir -E), cortisol, and prolactin (PRL) did not change under epidural analgesia per se, but after the start of laparoscopy, increased release of all these stress hormones was observed. General anesthesia per se increased the release of PRL, whereas the release of cortisol and ir -E decreased, probably due to the effects of fentanyl and thiopentone. During the stress attributed to laparoscopy, significantly more ir -E and cortisol was released under epidural than under general anesthesia, whereas the release of PRL was more significant under general anesthesia. These results show that neither mode of anesthesia prevented the stress response to laparoscopy. In the subsequent midluteal phase, the mean plasma level of progesterone and the mean progesterone-estradiol ratio were significantly greater in the epidural than in the general anesthesia group, suggesting that the mode of anesthesia may have an effect on the luteal phase. The significance of this difference on the conception rate remained unsolved, however.     

Effect of removal of cumulus cells from one-cell mouse embryos on in vitro development

The effects of the removal of cumulus cells from fertilized mouse oocytes (one-cell embryos) and the presence of streptomycin in culture medium on in vitro development were studied. Ham's F-10 medium with (0.075 g/liter) or without streptomycin was supplemented with human serum (15%). Cumulus-intact embryos were harvested from oviducts after mice were superovulated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Hyaluronidase (300 IU/ml) was used to remove the cumuli. Embryos were cultured (i) with cumulus/without streptomycin (n=238), (ii) with cumulus/with streptomycin (n=185), (iii) without cumulus/with streptomycin (n=210), and (iv) without cumulus/without streptomycin (n=218). Embryonic development was assessed 24, 96, and 120 hr after initiation of culture. Percentage two cells and percentage small or expanded blastocysts were not different (P>0.05) among experimental groups. Percentages hatched blastocysts for the four groups were (i) 36±8 and 54±7, (ii) 35±8 and 55±6, (iii) 19±5 and 42±6, (iv) 23±5 and 47±5 at 96 and 120 hr, respectively. Percentages all (small, expanded, and hatched combined) blastocysts were (i) 74±5 and 74±5, (ii) 74±9 and 72±5, (iii) 56±6 and 63±5, and (iv) 61±5 and 63±5 at 96 and 120 hr, respectively. A greater (P<0.05) percentage of embryos developed to blastocysts and hatched by 96 and 120 hr, when they were cultured with the cumulus intact. There was no effect of streptomycin on embryonic development. Cumulus cells may act as nurse cells during a critical stage of the development of the embryo.