Reduced allele dropout in single-cell analysis for preimplantation genetic diagnosis of cystic fibrosis

Background: For couples at risk of transmitting a known single-gene defect, preimplantation genetic diagnosis (PGD) allows the identification and transfer of only unaffected embryos followingin vitro fertilisation (IVF), single-cell biopsy at about the eight-cell stage, and genetic analysis by PCR. This technique therefore avoids the risk of terminating an affected pregnancy diagnosed later in gestation. Methods and Results: Using nested PCR, the F508 mutation causing cystic fibrosis can be detected in single cells and we previously reported successful PGD in a couple in whom both partners carry the F508 mutation. To date we have treated 12 couples in a total of 18 cycles. This resulted in five singleton births confirmed to be homozygous normal. Single blastomeres from disaggregated embryos which had not been transferred were analysed to confirm the original diagnosis and assess reliability in clinical practice. Amplification efficiency and accuracy were high, with blastomeres from embryos diagnosed as homozygous normal or affected. In a proportion of blastomeres from presumed carrier embryos, one of the parental alleles failed to amplify, apparently at random (allele dropout, ADO). A possible explanation is the relative inaccessibility of one of the target allele early in the PCR. To test this we have used single lymphocytes from F508 carriers and investigated the effects of various denaturation temperatures in the early cycles of amplification. Conclusion: Increasing the denaturation temperature reduced the rate of ADO without affecting amplification efficiency.Presented at the 5th Annual Meeting of the International Working Group on Preimplantation Genetics, Hamburg, Germany, June 28, 1995.     

The effect of activin-a on the development of mouse preimplantation embryos in vitro

Purpose: Our purpose was to clarify the involvement of transforming growth factor- (TGF-) family in the regulation of preimplantation embryo development. Methods: The effects of activin-A and TGF- on the rates of morula and blastocyst formations as well as on the cleavage velocity of a mouse two-cell embryo in vitro were analyzed. The gene expressions of these two growth factors in various developmental stages were also studied using RT-PCR. Results: Activin-A at a concentration of 0.2 ng/ml significantly stimulated not only the rate of morula formation but also the velocity of embryo cleavage, whereas no significant effect was found with TGF-. RT-PCR revealed that activin-A subunit mRNA, but not TGF- mRNA, was detected in preimplantation mouse embryo at any developmental stage. Conclusions: Activin-A plays an important role in the regulation of preimplantation mouse embryo development in an autocrine fashion.     

TNF-α in Pregnancy Loss and Embryo Maldevelopment: A Mediator of Detrimental Stimuli or a Protector of the Fetoplacental Unit?

Purpose : Tumor necrosis factor alpha (TNF-), a multifunctional cytokine, has been identified in the ovary, oviduct, uterus, and placenta, and is expressed in embryonic tissues. For many years TNF- was mainly considered to be a cytokine involved in triggering immunological pregnancy loss and as a mediator of various embryopathic stresses. However, data collected during the last decade has characterized TNF- not only as a powerful activator of apoptotic, but also antiapoptotic signaling cascades, as well as revealed its regulatory role in cell proliferation. This review summarizes and conceptualizes the studies addressing TNF--activated intracellular signaling and the possible functional role of TNF- in embryonic development. Methods : Studies addressing the role of TNF- in intercellular signaling, in vivo studies addressing the functional role TNF- in spontaneous and induced pregnancy loss, and studies addressing the role of TNF- in fetal malformations were reviewed. Comparative studies in TNF- knockout and TNF- positive mice were performed to evaluate embryonic death, structural anomalies in fetuses, the degree of apoptosis and cell proliferation, and the activity of molecules such as caspases 3 and 8, the NF-B, (RelA), IB in some target embryonic organs shortly after exposure to embryopathic stresses. Results : It is proposed that the possible essential function of TNF- may be to prevent the birth of offspring with structural anomalies. Conclusions : TNF- will boost death signaling to kill the embryo if initial events (damages) triggered by detrimental stimuli may culminate in structural anomalies, and stimulate protective mechanisms if the repair of these damages may prevent maldevelopment.     

Relationships Between Concentrations of Tumor Necrosis Factor-α and Nitric Oxide in Follicular Fluid and Oocyte Quality

Purpose: Our objective was to explain a relationshipbetween concentrations of tumor necrosis factor- (TNF-)and nitric oxide (NO) in follicular fluid, oocyte quality, andoutcomes of in vitro fertilization=nembryo transfer (IVF-ET). Methods: The concentrations of TNF- and NO weremeasured in 115 follicular fluid samples collected from 43patients undergoing IVF-ET program, due to tubalobstruction, some with endometriosis (8 patients) or hydrosalpinx(5 patients). A correlation of these factors concentrationsand the oocyte quality, the oocyte maturity, andinfertility-associated diseases was analyzed. Results: No correlation was found between concentrationsof NO and TNF- in follicular fluid. NO concentrations infollicular fluids were significantly higher in patients withendometriosis (P < 0.001) or hydrosalpinx (P < 0.01)compared to the patients with just tubal obstruction. FollicularNO concentration differences according to oocyte maturityand oocyte quality were not found. In contrast, TNF-concentrations in follicular fluids were significantly higher inpoor quality oocytes (P < 0.05) but were not associatedwith infertility-associated diseases, such as hydrosalphinxor endometriosis, and the oocyte maturity. No significantdifferences in follicular levels of NO and TNF- as well asIVF-ET parameters of pregnant and nonpregnant groupswere revealed. Conclusions: There is no significant correlation betweenthe concentrations of NO and TNF- in follicular fluid. NOlevels in follicular fluid are altered in infertility-associateddiseases. However, TNF- levels but not NO levels influenceoocyte quality. These results suggest that the production ofNO and TNF- in follicular fluid may be regulated viadifferent pathways and can be tempered withinfertility-associated diseases, thereby influencing oocyte qualitylocally.     

Reduced expression of alphavbeta3 integrin in the endometrium of unexplained infertility patients with recurrent IVF-ET failures: improvement by danazol treatment

Purpose : To determine whether there is any association between the expression of endometrial integrin v 3 and repeated IVF-ET failure and to examine the effect of danazol treatment on v 3 expression. Methods : This prospective study was performed using a semiquantitative immunohistochemical analysis on the staining intensity of v 3 in the mid-secretory endometria derived from 10 fertile women and 57 infertile patients with a history of repeated IVF-ET failures. Nine patients randomly selected from these 22 patients with unexplained infertility were then treated with oral danazol administration for 12 weeks and reexamined at the first mid-secretory phase after the danazol treatment. Result(s) : The levels of endometrial v 3 expression were lower in 22 patients with unexplained infertility than in the fertile control and 35 patients with explained infertility. The 9 patients treated with danazol showed a significant increase in the v 3 staining. Conclusion(s) : The significantly decreased expression of endometrial integrin v 3 suggested that functional, but not morphological, endometrial defect may be one of the causes for the patients with unexplained infertility. Danazol may have a therapeutic potential in improving endometrial function together with up-regulation of v 3.     

Clinical Assisted Reproduction: Altered Balance Between the 5α-Reductase and Aromatase Pathways of Androgen Metabolism During Controlled Ovarian Hyperstimulation with Human Menopausal Gonadotropins

Purpose: To evaluate androgen production and metabolism during controlled ovarian hyperstimulation. Methods: Five women, aged 33–42, were studied. All participants were undergoing controlled ovarian hyperstimulation with gonadotropin-releasing hormone agonist and human menopausal gonadotropins. Serum estradiol, estrone, androstenedione, testosterone, 3-androstanediol glucuronide, and sex hormone-binding globulin levels were measured at 6 time points during the cycle. Results: The levels of all steroids increased significantly from baseline during controlled ovarian hyperstimulation. Mean total testosterone levels increased from 0.29 ± 0.05 ng/mL to 0.58 ± 0.07 ng/mL after gonadotropin stimulation. Sex hormone-binding gonadotropin levels increased from 50 ± 16 nM to 73 ± 12 nM after gonadotropin stimulation. Estrone/androstenedione and estradiol/testosterone ratios, reflecting the aromatase pathway, increased whereas 3-androstanediol glucuronide/androstenedione and 3-androstanediol glucuronide/testosterone ratios, reflecting 5-reductase activity, decreased. Conclusions: Controlled ovarian hyperstimulation with human menopausal gonadotropins results in increased serum testosterone and androstenedione levels. Whereas there is an enhancement in androgen metabolism by aromatase, 5-reductase activity with regard to androgen metabolism is diminished.     

Allele Dropout in Polar Bodies and Blastomeres

Purpose: Because allele dropout (ADO) is frequently observed in single-cell polymerase chain reaction analysis, it is important to develop a method for efficient detection of ADO, in order to avoid possible misdiagnosis in preimplantation diagnosis. Methods: We introduced a simultaneous amplification of mutant genes and linked polymorphic markers, such as a 4-bp repeat (GATT) at the 3 end of intron 6 in the cystic fibrosis (CF) gene and a short tandem repeat at the 5 end of the -globin gene. Three types of single heterozygous cells were studied for the amplification of both alleles, including 150 blastomeres, 1615 fibroblasts, and 170 first polar bodies, obtained from patients at risk for having children with cystic fibrosis (delta F-508 mutation) or sickle cell disease. Results: ADO rates of as high as 33.3% for delta F-508 mutation and 22.8% for -globin gene were observed in single blastomeres, compared to 7.1 and 7.7% in single fibroblasts and 5.9 and 9.6% in first polar bodies, respectively. The application of simultaneous amplification of the above linked polymorphic markers allowed detection of more than half of the cases of ADO in blastomeres (19.4% for cystic fibrosis and 12.3% for -globin gene) and almost all ADOs in polar bodies, particularly when the two-step sequential analysis of the first and second polar body was applied in preimplantation diagnosis of single gene disorders. Conclusions: Simultaneous amplification of linked polymorphic markers in single-cell DNA analysis of single-gene defects is an efficient method for avoiding the risk of misdiagnosis in preimplantation diagnosis.     

Urinary excretion of 6-keto prostaglandin F1α in pre-eclampsia

Summary In order to investigate whether urinary excretion of prostaglandins (PG) is involved in the pathophysiology of pre-eclampsia, urinary immunoreactive 6-keto PGF₁ and TXB₂ were measured in normal and preeclamptic women by radio-immunoassay after extraction with Bond Elut column. Urinary levels of 6-keto PGF₁ and TXB₂ were expressed as ratio of urinary concentration of prostaglandin vs. creatinine (pg prostaglandin/mg creatinine; pg/mg crc.). Urinary excretion in normal pregnant and postpartum women were 211.2±33.8 and 160.1±9.1 pg/mg cre., respectively. In the pre-eclamptic group, urinary excretion of 6-keto PGF₁ was 105.3±28.2 pg/mg cre. in pregnancy and 99.0±12.5 pg/mg cre. in the postpartum period. Urinary excretion of 6-keto PGF₁ in the pre-eclamptic group was significantly lower (P<0.05) than in normal controls during pregnacy but not in the postpartum period. Urinary excretion of TXB₂ was not significantly different between the two groups. The urinary excretion of 6-keto PGF₁ was measured before and after the onset of pre-eclampsia in four cases of edema and weight gain of more than 500 g/week (group e), one case of proteinuria of more than 200 mg/dl with edema (group ep) and three cases of pre-eclampsia (group eph). The urinary excretion of 6-keto PGF₁ in these eight patients before onset of pre-eclampsia was slightly lower than of normal controls but not significantly so. In group eph, urinary excretion of PG was decreased after the onset of pre-eclampsia. These results provide further evidence of the involvement of PG in the pathophysiology of preeclampsia.     

The production of interleukin-1α immunoreactivity by human oviductal cells in a coculture system

Purpose: The aim of this study was to determine the concentration of interleukin-1 in human embryo culture medium with or without oviductal cell coculture and to correlate the interleukin-1 levels with pregnancy. Methods: Culture media from 32 in vitrofertilization and embryo transfer cycles were assayed for interleukin-1 by immunoassay technique. Human embryos were cultured in Earles' balanced salt solution supplemented with 15% preovulatory serum (sEBSS) in 16 of these cycles, while embryos in the rest of the cycles were cocultured with human oviductal cells in sEBSS. Results: Both sEBSS and spent sEBSS after embryo culture contained low or undetectable levels of interleukin-1 in the pregnant and nonpregnant cycles. On the other hand, oviductal cells significantly increased the amount of interleukin-1 immunoreactivity in the conventional culture medium or coculture medium (P<0.001, Mann-Whitney rank sum test). The concentrations of interleukin-1 in the spent sEBSS after oviductal cell culture and after coculture with human embryos were 1.5±1.0 and 1.3±0.9 pg/ml, respectively. There was no difference in the interleukin-1 concentration between the pregnant and the nonpregnant coculture cycles. Conclusions: These data showed that human oviductal cells produced interleukin-1 immunoreactivity in a coculture system. However, this production could not be used as a marker for successful embryo implantation.     

Polymorphisms for interleukin-1 beta (IL-1 beta)-511 promoter, IL-1 beta exon 5, and IL-1 receptor antagonist: nonassociation with endometriosis

Purpose: We aimed to investigate if interleukin-1 (IL-1) and IL-1 receptor antagonist (IL-1Ra) gene polymorphism could be used as markers of susceptibility in endometriosis. Materials and Methods: Women were divided into two groups: 1) endometriosis (n = 120); 2) nonendometriosis groups (n = 103). Polymorphisms for IL-1-511 promoter, IL-1 exon 5, and IL-1Ra were detected by polymerase chain reaction. Genotypes and allelic frequencies for these polymorphisms in both groups were compared. Results: Proportions of different IL-1 and IL-1Ra polymorphisms in both groups were nonsignificantly different. Proportions of C homozygote/heterozygote/T homozygote for IL-1-511 promoter in both groups were 1) 21.6/59.1/19.1% and 2) 26.2/50.5/23.3%. Proportions of E1 homozygote/heterozygote/E2 homozygote for IL-1 exon 5 in both groups were 1) 91.6/5/3.3% and 2) 95.15/4.85/0%. Allele I/II/IV/V for IL-1Ra in both groups were 1) 92.5/5.4/ 1.6/0.4% and 2) 95.1/3.9/1/0%. Conclusions: Association of endometriosis with IL-1-511 promoter, IL-1 exon 5, and IL-1 receptor antagonist gene polymorphisms doesn't exist. These polymorphisms are not useful markers for prediction of endometriosis susceptibility.     

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     

A controlled ovarian hyperstimulation regimen involving intermittent gonadotropin administration with a “short” protocol of gonadotropin releasing hormone agonist for in vitro fertilization

Purpose: To examine the effects of an intermittent injection regimen of exogenous gonadotropin for controlled ovarian hyperstimulation on follicular development and on in vitro fertilization (IVF) outcome, 120 women who were candidates for IVF received intermittent injection (II) or consecutive injection (CI) regimens with a short protocol (SP) or a long protocol (LP) of gonadotropin releasing hormone agonist (GnRHa). Pure follicle stimulating hormone (pFSH) was injected to the women in the II groups on the first, second, and fifth days of the stimulation cycle and every other day thereafter. The women in the CI groups received a daily injection of pFSH. An additional 16 patients who were treated with both II-SP and CI-LP were also analyzed. Results: Although the cancellation rate in the II-LP group was higher than those in the other groups, follicular development and IVF outcomes in the II-SP group were similar to those in the CI groups. The number of injections in the II-SP group was about half that in the CI groups. Conclusions: These results indicate that an intermittent pFSH injection regimen with a short protocol of GnRHa may be beneficial for patients in terms not only of being a less painful treatment but also causing less physical and mental stress than daily injections.     

Unilateral renal agenesis associated with ipsilateral blind vagina

Summary Activities of DNA polymerase and were assayed in crude extracts prepared from human placenta. Polymerase activity was high in early pregnancy but low during the 2nd trimester. Polymerase activity did not change significantly with gestational weeks. The increase in polymerase activity in early pregnancy may be closely related to mitosis at the time of placental formation; there might also be some type of accelerating factor for polymerase in early pregnancy.     

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.     

Increased Progesterone Secretion and 3β-Hydroxysteroid Dehydrogenase Activity in Human Cumulus Cells by Pregnenolone Is Limited to the High Steroidogenic Active Cumuli

Purpose: Several reports imply that lower progesteronesecretion by cumulus-oocyte complexes (COCs) isassociated with lower fertilization in the corresponding oocyte.The possible role of progesterone in oocyte fertilization inhumans was studied using two approaches: (a) increasingthe total progesterone secretion by culturing more than oneCOC per dish; and (b) increasing the cumulus cell progesterone secretion by providing pregnenolone as a substrate. Methods: Mature COCs were cultured individually orcocultured in groups. Oocyte fertilization and progesteronesecretion were tested after 20 hr and 3 days in culture, respectively.The cumuli from individually plated COCs were cultured inthe absence of oocyte for an additional 3 days in order totest the effects of pregnenolone on progesterone secretionand the 3-hydroxysteroid dehydrogenase (3-HSD)activity. A comparable study with pregnenolone was performedon the corresponding granulosa-lutein cells. Results: Increasing the number of COC to two instead ofone led to a significant increase in both fertilization rateand progesterone secretion. The addition of pregnenoloneduring days 3–6 increased significantly both progesteronesecretion and 3-HSD activity. Comparable results wereobserved in granulosa-lutein cells subjected to pregnenolonetreatment. Following the first 3 days culture, cumulus masseswere categorized as secreting high or low progesteronelevels. Adding pregnenolone had a greater effect on bothprogesterone secretion and 3-HSD activity in thehigh-progesterone-secreting cumuli. Conclusions: Addition of pregnenolone increased progesterone secretion and 3-HSD more efficiently in thehigher-progesterone-secreting cumuli. Coculture of two COCsinstead of one led to a higher fertilization rate and greaterprogesterone secretion.     

Reliability of polymerase chain reaction (PCR) analysis of single cells for preimplantation genetic diagnosis

Purpose We investigated the reliability of polymerase chain reaction (PCR) genotype analyses performed on single cells for the purposes of preimplantation genetic analysis.Methods We performed blind analysis of 130 single skin fibroblasts heterozygous for the -F508 mutation in the cystic fibrosis transmembrane regulator (CFTR) gene and 73 single skin fibroblasts from an individual heterozygous for the Xbapolymoporphic site of the Factor VIII gene.Results Amplification was successful for 116 cells and 52 cells respectively and in all but one case (a CFTR analysis) both alleles were amplified. The incidence of diagnostic error was 1 out of 203 analyses or 0.0043. We conclude that PCR is a reliable method for determining the genotype of single cells for the purposes of preimplantation genetic analysis.     

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.     

Polymorphism in the tumor necrosis factor-alpha gene in women with preeclampsia

Purpose : We determined whether genetic variability in the gene encoding for tumor necrosis factor- (TNF-) contributes to individual differences in susceptibility to the development of preeclampsia. Methods : The study involved 133 preeclamptic and 115 healthy control pregnant women who were genotyped for C-850T polymorphism in the TNF- gene promoter. Chi-square analysis was used to assess genotype and allele frequency differences between preeclamptic women and controls. Results : A significantly different genotype distribution of C-850T polymorphism was observed between the two groups, with the frequency of the variant T allele being significantly reduced in the preeclamptic group (4.5%) when compared with the control group (9.6%) (P = 0.03; OR = 0.45, 95% CI = 0.22–0.92). Accordingly, the odds ratio for preeclampsia associated with the pooled TT and CT genotypes was 0.367 (P = 0.02; 95% CI = 0.159–0.847). Conclusions : The T allele of the TNF- gene may modify individual preeclampsia risk, being protective against the development of the complication.     

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.