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PubMed1997

Human Fallopian tube epithelial cell co-culture increases fertilization rates in male factor infertility but not in tubal or unexplained infertility.

In order to investigate the effect of human Fallopian tube epithelial cell co-culture on fertilization and cleavage rates in tubal, male and unexplained infertility, oocytes collected from 91 patients were randomized to wells containing Fallopian tube epithelial cell monolayers or conventional culture medium, and inseminated with spermatozoa. Fertilization and cleavage were assessed at 18 and 52 h, respectively. Co-culture significantly increased the fertilization rates over the control values in male infertility (41.67 versus 23.43%, P = 0.00005), but not in tubal infertility (69.33 versus 67.93%) or unexplained infertility (65.93 versus 54.36%). Cleavage rates were not different in co-culture and conventional in-vitro fertilization systems in any of the infertility subgroups. The number of blastomeres was significantly higher in the co-culture group on the day of embryo transfer (3.63 +/- 1.12 versus 3.04 +/- 1.26, P < 0.001). Pregnancy rates were similar in all infertility subgroups. There was no significant association between the number of co-cultured embryos transferred and the pregnancy, abortion and multiple pregnancy rates. It was concluded that human Fallopian tube epithelial cell co-culture clearly improves fertilization rates in male infertility but not in tubal or unexplained infertility. Improved fertilization rates in co-culture may be due to positive effect of co-culture on impaired sperm function.

Blastomeres↗
PubMed1998

Fertilization and embryo development to blastocysts after intracytoplasmic sperm injection in the rhesus monkey.

Notwithstanding the thousands of seemingly healthy children born after intracytoplasmic sperm injection (ICSI), it is not yet possible to conclude absolutely that the ICSI procedure might induce some altered development or that the ICSI protocol might not be improved even further. To address this in a clinically relevant system, the developmental potential of rhesus monkey embryos produced by ICSI is reported. Oocytes collected by laparoscopy from gonadotrophin-stimulated fertile females were fertilized by ICSI using spermatozoa obtained from fertile males by electro-ejaculation. Neither sperm immobilization prior to injection nor an additional chemical stimulus were necessary to achieve oocyte activation and pronuclear formation. Survival and activation of the injected oocytes were judged by the extrusion of the second polar body. Successful fertilization was confirmed by the presence of two pronuclei within 12 h post-ICSI. Some oocytes were fixed and processed for the detection of microtubules and chromatin. Fluorescent labelling revealed that by 12 h post-ICSI the male and female pronuclei were closely apposed and eccentrically positioned within a large microtubule aster. ICSI resulted in a 76.6 +/- 14.9% fertilization rate. First cleavage was completed within 24 h post-ICSI. Two-cell ICSI embryos were co-cultured in CMRL medium on a buffalo rat liver cell monolayer until the hatched blastocyst stage. Oocytes collected laparoscopically from stimulated monkeys can be fertilized by ICSI and will complete preimplantation embryo development in vitro demonstrating that the rhesus monkey is an excellent preclinical model for examining and understanding many aspects of human ICSI.

Animals↗
PubMed1999

Controlled comparison of conventional in-vitro fertilization and intracytoplasmic sperm injection in patients with asthenozoospermia.

A controlled comparison between conventional in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) has been carried out for patients with </=5% rapid progressive (type A) spermatozoa in their fresh semen. Twenty couples were allocated to the study. All semen samples fulfilled the minimum criteria to be allocated to the conventional IVF programme. The two pronuclear (2PN) fertilization rate per retrieved cumulus-oocyte complex was significantly higher after ICSI (63.4%) than after conventional IVF (22.9%), with complete fertilization failure after IVF in 10 out of the 20 cycles. Embryo quality was similar for both treatments. Sixteen patients received only ICSI embryos, two patients only IVF embryos and two patients received IVF and ICSI embryos. The ongoing clinical pregnancy rate was 45%, the implantation rate was 37%. Comparison of the characteristics of patients/cycles with and without 2PN fertilization revealed a higher proportion of progressively motile spermatozoa in the prepared sperm fraction for the group of patients with fertilization after conventional IVF. It can be concluded from this study that absence of or an extremely low proportion of rapid progressive motility in fresh semen indicates a high risk of complete fertilization failure with conventional IVF.

Embryo Implantation↗
PubMed2000

Computer-assisted semen analysis parameters as predictors for fertility of men from the general population. The Danish First Pregnancy Planner Study Team.

The predictive value of sperm motility parameters obtained by computer-assisted semen analysis (CASA) was evaluated for the fertility of men from general population. In a prospective study with couples stopping use of contraception in order to try to conceive, CASA was performed on semen samples from 358 men. A recently developed CASA system, Copenhagen Rigshospitalet Image house sperm Motility Analysis System (CRISMAS) was used for assessment of motility parameters. This system has an editing function which allows correction of tracks made by the computer. Probably due to this function, the concentration assessment made by CRISMAS was very close to that made by the technician (median difference <5%) in all concentration ranges. Correlation between CASA parameters and fertility of normal couples (measured as probability of achieving pregnancy) was examined by the Cox regression model. In univariate models ln(sperm concentration) [beta = 0.331, risk ratio (RR) = 1.392, P = 0.0001], ln(total sperm count) (beta = 0.252, RR = 1.286, P = 0.0007) and percentage motile spermatozoa (beta = 0.014, RR = 1.014, P = 0.0004) were most significant predictors for fertility. In a multivariate analysis ln(sperm concentration) (beta = 0.268, RR = 1.307, P = 0.0016) and percentage motile spermatozoa (beta = 0.010, RR = 1.010, P = 0.011) but even more significantly the combined parameter, ln(concentration of motile spermatozoa) (beta = 0.329, RR = 1.389, P = 0.0001), were the only parameters of predictive value for fertility of men in the general population. In conclusion, these parameters obtained by CASA measurements can be used for prediction of fertility potential in normal men. This appears to be the first study showing the value of CASA in prediction of fertility in the general male population.

Adult↗
PubMed2002

A comparison of mitochondrial and nuclear DNA status in testicular sperm from fertile men and those with obstructive azoospermia.

BACKGROUND: Mitochondria are vital to sperm as their motility powerhouses. They are also the only animal organelles with their own unique genome; encoding subunits for the complexes required for the electron transfer chain. METHODS: A modified long PCR technique was used to study mitochondrial DNA (mtDNA) in ejaculated and testicular sperm samples from fertile men undergoing vasectomy (n = 11) and testicular sperm from men with obstructive azoospermia (n = 25). Nuclear DNA (nDNA) fragmentation was measured by an alkaline gel electrophoresis (comet) assay. RESULTS: Wild-type mtDNA was detected in only 60% of fertile men's testicular sperm, 50% of their ejaculated sperm and 46% of testicular sperm from men with obstructive azoospermia. The incidence of mitochondrial deletions in testicular sperm of fertile and infertile men was not significantly different, but the mean size of the deletions was significantly less in testicular sperm from fertile men compared with men with obstructive azoospermia (P < 0.02). NDNA fragmentation in testicular sperm from fertile men and men with obstructive azoospermia was not significantly different. CONCLUSION: Multiple mtDNA deletions are common in testicular and ejaculated sperm from both fertile and infertile men. However, in males with obstructive azoospermia, the mtDNA deletions in testicular sperm are of a larger scale.

Base Sequence↗
PubMed2006

Psychological distress by type of fertility barrier.

BACKGROUND: We examined fertility-specific distress (FSD) and general distress by type of fertility barrier (FB). METHODS: In a random sample telephone survey, 580 US women reported their fertility intentions and histories. Six groups of women were identified: (i) no FBs, (ii) infertile with intent, (iii) infertile without intent, (iv) other fertility problems, (v) miscarriages and (vi) situational barriers. Multiple regression analyses were used to compare groups with FBs. RESULTS: Sixty-one percent reported FBs and 28% reported an inability to conceive for at least 12 months. The infertile with intent group had the highest FSD, which was largely explained by (a) self-identification as infertile and (b) seeking medical help for fertility. The no FB group had a mean Center for Epidemiological Studies Depression scale score above the commonly used cut-off of 16, although 23% of the women with FBs did score above 16. CONCLUSIONS: FBs are common. Self-identification as infertile is the largest source of FSD. More women with FBs had elevated general distress than women without FBs; mean general distress was below 16 for all FB groups. It may be that, for some women (even those with children), FBs can have lasting emotional consequences, but many women do heal from the emotional distress that may accompany fertility difficulties.

Abortion, Spontaneous↗
PubMed2006

Loss of ovule identity induced by overexpression of the fertilization-related kinase 2 (ScFRK2), a MAPKKK from Solanum chacoense.

In order to gain information about protein kinases acting during plant fertilization and embryogenesis, a reverse genetic approach was used to determine the role of protein kinases expressed in reproductive tissues. Two cDNA clones named ScFRK1 and ScFRK2 (Solanum chacoense fertilization-related kinase 1 and 2) were isolated from an expressed sequence tag (EST) library normalized for weakly expressed genes in fertilized ovaries. These showed significant sequence similarities to members of the mitogen-activated protein kinase kinase kinase (MAPKKK) family. RNA gel blot and RNA in situ hybridization analyses confirmed the strong up-regulation of ScFRK2 in ovules after fertilization. In addition, ScFRK2 mRNAs accumulate during early ovule development in the megasporocyte and in the integument of developing ovules. Overexpression of ScFRK2 led to the production of fruits with a severely reduced number of seeds. The seeds that were produced also exhibited developmental retardation. Analysis of ovaries prior to fertilization showed that the seedless phenotype was caused by a homeotic conversion of ovules into carpel-like structures. The present observations are consistent with the role of ScFRK2 in pre- and post-fertilization events. Furthermore, overexpression of ScFRK2 led to changes in the expression of the class D floral homeotic gene ScFBP11, suggesting that the ScFRK2 kinase may interact, directly or indirectly, with the FBP7/11 pathway that directs establishment of ovule identity.

Amino Acid Sequence↗
PubMed2005

Temporary developmental arrest after storage of fertilized mouse oocytes at 4 degrees C: effects on embryonic development, maternal mRNA processing and cell cycle.

The aim of this study was to examine whether fertilized mouse oocytes can survive after short-term incubation (for 6-48 h) at 4 degrees C. When fertilized oocytes of ICR and C57BL/6 (B6) strain were incubated at 4 degrees C and returned to normal culture conditions (37 degrees C), development of these 4 degrees C-treated embryos for up to 12 h (for ICR) to blastocyst stage did not differ from that of untreated oocytes. Even 4 degrees C-treated embryos for 48 h developed to blastocysts at relatively good rates (33.3% for ICR and 50.8% for B6). The in vivo development of 4 degrees C-treated embryos for 12, 24 and 36 h to fetal stage was similar to that of untreated ones. BrdU labelling assay revealed temporary cessation of DNA replication in 4 degrees C-treated fertilized oocytes. Post-fertilization events including cytoplasmic polyadenylation of maternal mRNAs, mRNA degradation of a cell cycle-related gene and elevated mRNA expression of zygotic gene activation-related genes were temporarily suppressed in 4 degrees C-treated embryos. These findings indicate that 4 degrees C-treatment of fertilized murine oocytes results in temporary cessation of molecular events. We also show that 4 degrees C-treated fertilized oocytes for 12 h can be used for preparation of transgenic mice.

Animals↗
PubMed1996

In-vitro fertilization: the experience of treatment, pregnancy and delivery.

The present study compares the evidence of pregnancy and delivery among in-vitro fertilization (IVF) parents (45 couples), other formerly infertile parents (35 couples) and fertile parents (35 couples). All deliveries concerned primaparous women and singleton births. In addition, the burden of fertility treatments was investigated. Results show that the psychological burden of the treatments exceeds the physical burden. Fertility treatments were judged very worthwhile. Complications during pregnancy were more frequently reported by IVF mothers and other initially infertile mothers than by fertile mothers. However, controlling for the older age in both formerly infertile groups (IVF and non-IVF), no significant difference was found. No differences appeared regarding the evaluation of the development of the delivery. IVF parents and other infertile parents evaluated the pregnancy as more stressful than fertile parents. However, mothers experienced their delivery as more exceptional, and fathers experienced the pregnancy as more exceptional. In addition, IVF fathers enjoyed the pregnancy more than fathers from the other groups.

Female↗
PubMed1995

Polyamines may increase the percentage of in-vitro fertilized murine oocytes that develop into blastocysts.

This study was designed to investigate whether the polyamines spermine, spermidine and putrescine can prevent the arrest of development of in-vitro fertilized murine oocytes from CD1 strain mice. Development in M16 or Brinster's medium with or without polyamine supplementation was assessed and the ability of blastocysts to attach and grow out on laminin was compared with in-vivo-generated embryo blastocysts. Oocytes from old (20 weeks) females showed lower rates of fertilization and subsequent development in comparison with oocytes from young (6-8 weeks old) females, but in both cases < 5% of in-vitro fertilized oocytes achieved the blastocyst stage. In-vitro fertilized young oocytes showed enhanced development to the blastocyst stage with the addition of the polyamine putrescine, although lesser enhancement was seen with spermine and spermidine; this effect was only evident when the oocytes were grown in Brinster's medium, reflecting the need for a particular set of culture conditions for growth-enhancing agents to be effective. Blastocysts generated in putrescine-supplemented Brinster's medium were able to implant on laminin-coated surfaces in vitro and to respond to the cytokines colony-stimulating factor-1, epidermal growth factor and platelet-derived growth factor with increased trophoblast outgrowth to a greater degree than in-vivo-generated blastocysts. Murine in-vitro fertilized embryo development was not enhanced by co-culture with Vero cells. Putrescine was the most effective polyamine, enhancing the in-vitro fertilized oocyte development to blastocysts in a permissive environment.

Animals↗
PubMed1995

Does zona pellucida thickness influence the fertilization rate?

In human in-vitro fertilization (IVF), the cumulus oophorus is routinely removed to assess fertilization and hence the thickness of the zona pellucida is measurable. This study aimed to measure the thickness of the zona pellucida and to assess its influence on fertilization rate in IVF programmes. The zona pellucida thickness varies from 10 to 31 microns with a mean of 17.5 microns. One-way analysis of variance revealed that in IVF trials performed with normal semen, the zona pellucida of fertilized oocytes (16.6 +/- 3.2 microns) was significantly thinner than the zona pellucida of unfertilized oocytes (18.9 +/- 4.0 microns; P < 0.001). As measured on micro-injected oocytes, the zona pellucida thickness did not change between ovulation and 16-20 h after fertilization. Zona pellucida thickness was not related to ooplasm diameter. In conclusion, zona pellucida thickness appears to be an additional factor that should be taken into account when interpreting the fertilization rate. Zona pellucida thickness influences sperm penetration, even when the spermatozoa are considered normal. From a clinical point of view, a thick zona pellucida (> or = 22 microns) could be an indicator for the use of micro-injection procedures.

Adult↗
PubMed1995

A new predictive test for in-vitro fertilization based on the induction of sperm acrosome reaction by N-acetylglucosamine-neoglycoprotein.

Neoglycoproteins with N-acetylglucosamine residues (BSA-GlcNAc) induced specifically the acrosome reaction (AR) in human spermatozoa. Our objective was to investigate the relationship between this phenomenon and the invitro fertilization (IVF) rate. Sperm suspensions from IVF protocols were incubated with BSA-GlcNAc (t), using calcium ionophore (i) or medium alone (c) as positive or negative controls. When the normalized AR percentage ratio (STIM) (% ARt-%ARc):(%ARi-%ARc) was compared with fertilization rate in 31 couples from our IVF programme, a positive correlation was found (r = 0.46, P < 0.01). The fertilization rate in patients with STIM > or = 0.2 was higher than in non-responders (STIM < 0.2); 72 +/- 7% compared with 5 +/- 3%. The overall predictive value of this test for adequate fertilization rate (> 30%) was 87%, sensitivity 91% and specificity 78%. False positives were 9% and false negatives 22%. For successful fertilization rates (> 60%), the results were: overall predictive value, 84%; sensitivity 100%; specificity 64%. False positives were 23% and no false negatives were found. The results indicated that the induction of AR in human spermatozoa by GlcNAc-neoglycoproteins could be used to predict their fertilizing ability in vitro.

Acetylglucosamine↗
PubMed1987

The occurrence, recognition and developmental fate of pseudo-multipronuclear eggs after in-vitro fertilization of human oocytes.

The presence of three or more presumed pronuclei in 27 of 884 (3%) in-vitro fertilized human eggs suggested the occurrence of polyspermic fertilization. Detailed examination by differential interference contrast microscopy indicated that approximately 40% of presumed multipronuclear eggs were normally fertilized but contained a cytoplasmic vacuole(s) (pseudo-pronucleus) of pronuclear dimension and gross morphology. Closely timed observations of presumed multipronuclear eggs revealed that pseudo-pronuclei (PPN) can co-migrate and become juxtaposed with true pronuclei, thus presenting the impression of an aberrant fertilization. Fluorescent probe and electron microscopic analyses demonstrated that PPN are enclosed by a plasma membrane and contain neither DNA nor nucleoli. PPN can develop either in the mature oocyte or the newly fertilized egg. Pseudo-multipronuclear eggs progress through the preimplantation stages in an apparently normal fashion and, as demonstrated by one birth and two ongoing pregnancies, are developmentally viable. Criteria for the unambiguous designation of pseudo-multipronuclear eggs are based on: the absence of normal morphodynamic changes associated with pronuclear development; and the failure of nucleoli to appear during the perisyngamic stage. The findings strongly suggest that pseudo-multipronuclear human eggs may represent a class of morphological variants of normally fertilized and developmentally competent eggs.

Cell Nucleolus↗
PubMed1988

Factors associated with accidental fractures of the zona pellucida and multipronuclear human oocytes following in-vitro fertilization.

A total of 101 multipronuclear oocytes (7.4% of fertilizations) were retrospectively identified in this in-vitro fertilization programme. The use of a manual syringe suction system, instead of an electric pump, to aspirate follicles, was associated with a significant increase in the proportion of oocytes with fractured zonae pellucidae (P less than 0.001), a lower normal fertilization rate (P less than 0.05) and a higher proportion of multipronuclear fertilizations (P less than 0.001). Irrespective of the mode of follicular aspiration, significantly more multipronuclear fertilizations occurred following stimulation with a combination of clomiphene and human menopausal gonadotrophin, than after clomiphene alone (P less than 0.05). It was concluded that the aspiration pressures, created by syringe suction, were more likely to rupture the zona pellucida of some oocytes, while in others it predisposed to an increased multipronuclear fertilization rate.

Female↗
PubMed1989

Human sperm-zona pellucida binding, sperm characteristics and in-vitro fertilization.

The number of sperm bound to the zona pellucida (ZP) were counted on 660 oocytes that failed to fertilize in vitro from 162 patients. Oocytes from clutches in which some fertilized had significantly more sperm bound to the ZP than did those from clutches in which all oocytes failed to fertilize. Oocytes from patients in whom all were not fertilized and no sperm bound to the ZP were able to bind normal fertile donor sperm after storage in ammonium sulphate solution. The number of sperm bound to the ZP was significantly related to the proportion of sperm with normal morphology and normal intact acrosomes in semen, sperm concentration inseminated, sperm motility and viability. The number of sperm bound to the ZP, sperm normal morphology, diagnosis of male infertility and sperm concentration in semen were significantly related to the fertilization rate by logistic regression analysis. Thus the number of sperm bound to the ZP is an indicator for IVF success and low binding appears to be more frequently associated with sperm defects than oocyte defects.

Adult↗
PubMed1989

Steroid production by the cumulus: relationship to fertilization in vitro.

Insemination media were collected from 92 follicles of 14 patients stimulated to undergo oocyte retrieval for in-vitro fertilization. Levels of progesterone and oestradiol in the insemination drops were assayed, corrected for carry-over from follicular fluid and volume and expressed as production per microgram of protein in the cumulus. Significantly higher progesterone production per unit protein was associated with oocytes which fertilized in vitro (P less than 0.02). Oocytes fertilizing with subsequent fragmentation or degeneration showed progesterone levels significantly higher than oocytes fertilizing normally (P less than 0.05). Polyspermic oocytes (n = 3) were associated with very high levels of progesterone production but were not significantly different due to the low numbers. Oestradiol production per unit protein was significantly greater in oocytes which fertilized normally than in those which degenerated (P less than 0.05). The protein content of cumuli whose oocytes fertilized appeared to be significantly lower than those which did not (P less than 0.05). These results probably reflect the maturity of the follicle, although direct actions of cumulus products upon the gametes cannot be ruled out.

Cells, Cultured↗
PubMed1991

Microsurgical fertilization and teratozoospermia.

Semen parameters were correlated with the outcome of partial zona dissection (PZD) in 42 couples with male factor infertility. Although fertilization rates were reduced, 12% of the embryos implanted following replacement. Spermatozoa from teratozoospermic sperm populations were able to fuse with oocytes following zona penetration through the artificial gaps. PZD followed by insemination with less than 5% normal spermatozoa led to 20 embryos which, upon replacement, did not implant. Motility and sperm count were not clearly correlated with the outcome of PZD and are therefore less useful indicators for patient selection. Teratozoospermic patients who previously failed to fertilize were compared to a group of similar patients who had not attempted IVF before. Although fertilization was significantly improved in first-time patients, 41% of the patients whose spermatozoa were initially unable to fertilize had at least one embryo when PZD was performed. Several pregnancies were established in this group. Subzonal sperm insertion (SZI) and PZD were compared in 19 patients using sibling oocytes. A significant fraction of spermatozoa from infertile men were able to fuse with the oolemma when directly inserted into the perivitelline area. Using a sucrose solution to shrink the ooplasm, only 1% of the oocytes were damaged during SZI. Monospermic fertilization rates following PZD and SZI were 15 and 16%, respectively. Both micromanipulation methods were successful in most patients. However, in two small groups of patients, only one technique resulted in fertilization.

Female↗
PubMed1991

Fertilization and ageing processes in non-divided human oocytes after GnRHa treatment: an analysis of individual oocytes.

Some human oocytes cultured together with spermatozoa for in-vitro fertilization (IVF) do not subsequently divide. The arrest of the fertilization process at different moments during development may provide information about the cause of fertilization failure. Oocytes which subsequently divide are transferred 48 h after insemination; when oocytes do not divide, ageing processes can be observed. Therefore these oocytes are interesting material in which to observe both fertilization and ageing. Our study concerns 72 undivided human oocytes 0, 48 or 72 h post-insemination. DNA of the oocyte and spermatozoa was visualized by the DNA fluorescent dye Hoechst 33342. Living oocytes were observed in toto by fluorescence and bright field microscopy which allowed nuclear and pronuclear membranes to be discerned. Oocytes were subsequently fixed and sectioned for bright field microscopy. Both techniques allowed parallel observations. Oocytes at various stages of fertilization are described: sperm penetration in both mature and immature oocytes, decondensation of sperm-heads, premature condensation of male chromatin, polyspermy and pronucleus formation. Typical ageing processes such as the centripetal migration of the metaphase II chromosomes, the formation of a restitution nucleus and the lagging of chromosomes within a metaphase spindle are observed. DNA fluorescence appears to be a quick, easy and valuable means to analyse fertilization and its failure.

Adult↗