Fertilization Of Human Oocytes Research Articles

Investigation of ADAMTS-1, BAX and IL-10 Expressions in Granulosa Cells Determinanting Fertilization Success in IVF

Granulosa cells (GCs) are essential components of follicles and play a role in regulating follicle development. The aim of this study was to investigate certain cellular components involved in the proliferation, differentiation and functional characteristics of granulosa cells in the success of fertilization of human oocytes during invitro fertilization (IVF) via immunohistochemical techniques. In this study, 30 patients who were diagnosed as primary or secondary infertility, polycystic ovary syndrome in the IVF center of Memorial Hospital, Department of Obstetrics and Gynecology were included. The amount of Anti Müllerian Hormone (AMH) in blood and granulosa cell diameter and cell core diameter were measured in 20 cells collected from each patient. In addition, degeneration scoring and BAX, ADAMTS-1, IL-10 expressions in granulosa cells were evaluated (p <0.01). It was thought that apoptosis induced by human GCs might be an indicator of egg quality. Moderate expression of ADAMTS-1 was thought to be related to failure of ovulation, deterioration of oocyte quality and decreased fertilization rate. This decrease in AMH levels may be associated with defects in granulosa cells. Therefore, significantly lower AMH secretion and increase in IL10 expression levels in healthy people can be explained by the increase of granulocyte cells.

Preventing Common Hereditary Disorders through Time-Separated Twinning

Biomedical advances have led to a relaxation of natural selection in the human population of developed countries. In the absence of strong purifying selection, spontaneous and frequently deleterious mutations tend to accumulate in the human genome and gradually increase the genetic load; that is, the frequency of potentially lethal genes in the gene pool. Gradual increase in incidence of many complex disorders suggests deleterious impact of the genetic load on human well-being. Recent advances in in vitro fertilization (IVF) combined with artificial twinning and transgenerational embryo cryoconservation offer the possibility of preventing significant accumulation of genetic load and reducing the incidence of hereditary disorders. Many complex diseases such as type 1 and 2 diabetes, autism, bipolar disorder, allergies, Alzheimer’s disease, and some cancers show significantly higher concordance in monozygotic (MZ) twins than in fraternal twins (dizygotic, DZ) or parent-child pairs, suggesting their etiology is strongly influenced by genetics. Preventing these diseases based on genetic data alone is frequently impossible due to the complex interplay between genetic and environmental factors. We hypothesize that the incidence of complex diseases could be significantly reduced in the future through a strategy based on time-separated twinning. This strategy involves the collection and fertilization of human oocytes followed by several rounds of artificial twinning. If preimplantation genetic screening (PGS) reports no aneuploidy or known Mendelian disorders, one of the MZ siblings would be implanted and the remaining embryos cryoconserved. Once the good health of the adult MZ sibling(s) is established, subsequent parenthood with the cryoconserved co-twins could substantially lower the incidence of hereditary disorders with complex etiology and virtually eradicate simple Mendelian disorders. The proposed method of artificial twinning has the potential to alleviate suffering and reduce the negative social impact induced by dysgenic effects associated with known and unknown genetic factors. Time-separated twinning has the capacity to prevent further accumulation of the genetic load and to provide source of isogenic embryonic stem cells for future regenerative therapies.

A role for retinoids in human oocyte fertilization: regulation of connexin 43 by retinoic acid in cumulus granulosa cells.

Retinoids are essential for ovarian steroid production and oocyte maturation in mammals. Oocyte competency is known to positively correlate with efficient gap junction intercellular communication (GJIC) among granulosa cells in the cumulus-oocyte complex. Connexin 43 (C x 43) is the main subunit of gap junction channels in human cumulus granulosa cells (CGC) and is regulated by all-trans retinoic acid (ATRA) in other hormone responsive cell types. The objectives of this study were to quantify retinoid levels in human CGC obtained during IVF oocyte retrievals, to investigate the potential relationship between CGC ATRA levels and successful oocyte fertilization, and to determine the effects of ATRA on C x 43 protein expression in CGC. Results showed that CGC cultures actively metabolize retinol to produce ATRA. Grouped according to fertilization rate tertiles, mean ATRA levels were 2-fold higher in pooled CGC from women in the highest versus the lowest tertile (P < 0.05). ATRA induced a rapid dephosphorylation of C x 43 in CGC and granulosa cell line (KGN) cultures resulting in a >2-fold increase in the expression of the functional non-phosphorylated (P0) species (P < 0.02). Similar enhancement of P0 by ATRA was shown in CGC and KGN cultures co-treated with LH or hCG which, by themselves, enhanced the protein levels of C x 43 without altering its phosphorylation profile. Correspondingly, the combination of ATRA+hCG treatment of KGN caused a significant increase in GJIC compared with single agent treatments (P < 0.025) and a doubling of GJIC from that seen in untreated cells (P < 0.01). These findings indicate that CGC are a primary site of retinoid uptake and ATRA biosynthesis. Regulation of C x 43 by ATRA may serve an important role in folliculogenesis, development of oocyte competency, and successful fertilization by increasing GJIC in CGC.

Oocyte cryopreservation and in vitro culture affect calcium signalling during human fertilization

What are the precise patterns of calcium oscillations during the fertilization of human oocytes matured either in vivo or in vitro or aged in vitro and what is the effect of cryopreservation? Human oocytes matured in vivo exhibit a specific pattern of calcium oscillations, which is affected by in vitro maturation, in vitro ageing and cryopreservation. Oscillations in cytoplasmic calcium concentration are crucial for oocyte activation and further embryonic development. While several studies have described in detail the calcium oscillation pattern during fertilization in animal models, studies with human oocytes are scarce. This was a laboratory-based study using human MII oocytes matured in vivo or in vitro either fresh or after cryopreservation with slow freezing or vitrification. Altogether, 205 human oocytes were included in the analysis. In vivo and in vitro matured human oocytes were used for this research either fresh or following vitrification/warming (V/W) and slow freezing/thawing (F/T). Human oocytes were obtained following written informed consent from patients undergoing ovarian hyperstimulation. For the calcium pattern analysis, oocytes were loaded with the ratiometric calcium indicator fluorescent dye Fura-2. Following ICSI using sperm from a single donor, intracellular calcium was measured for 16 h at 37°C under 6% CO(2). The calcium oscillation parameters were calculated for all intact oocytes that showed calcium oscillations and were analyzed using the Mann-Whitney U-test. Human in vivo MII oocytes display a specific pattern of calcium oscillations following ICSI. This pattern is significantly affected by in vitro ageing, with the calcium oscillations occurring over a longer period of time and with a lower frequency, shorter duration and higher amplitude (P < 0.05). In vitro matured oocytes from the GV and MI stage exhibit a different pattern of calcium oscillations with calcium transients being of lower frequency and shorter duration compared with in vivo matured MII. In MI oocytes that reached the MII stage within 3 h the calcium oscillations additionally appear over a longer period of time (P < 0.05). In vivo MII oocytes show a different calcium oscillation pattern following V/W with calcium oscillations occurring over a longer period of time, with a higher amplitude and a lower frequency (P < 0.05). In vitro matured oocytes, either from the GV or the MI stage, also display an altered pattern of calcium oscillations after V/W and the parameters that were similarly affected in all these oocyte groups are the frequency and the amplitude of the calcium transients. Slow freezing/thawing differentially affects the calcium oscillation pattern of in vitro matured and in vitro aged oocytes. The relationship between a specific pattern of calcium oscillations and subsequent human embryonic development could not be evaluated since the calcium indicator used and the high-intensity excitation light impair development. Furthermore, all oocytes were derived from stimulated cycles and immature oocytes were denuded prior to in vitro maturation. Our data show for the first time how calcium signalling during human fertilization is affected by oocyte in vitro maturation, in vitro ageing as well as V/W and slow freezing/thawing. The analysis of calcium oscillations could be used as an oocyte quality indicator to evaluate in vitro culture and cryopreservation techniques of human oocytes. This work was supported by a clinical research mandate from the Flemish Foundation of Scientific Research (FWO-Vlaanderen, FWO09/ASP/063) to F.V.M, a fundamental clinical research mandate from the FWO-Vlaanderen (FWO05/FKM/001) to P.D.S and a Ghent University grant (KAN-BOF E/01321/01) to B.H. The authors have no conflict of interest to declare.

Shake, rattle and roll: bringing a little rock to the IVF laboratory to improve embryo development

Shake, rattle and roll: bringing a little rock to the IVF laboratory to improve embryo development

TUNEL analysis of DNA fragmentation in mouse unfertilized oocytes: The effect of microorganisms within human follicular fluid collected during IVF cycles

Recently we reported the presence of bacteria within follicular fluid. Previous studies have reported that DNA fragmentation in human spermatozoa after in vivo or in vitro incubation with bacteria results in early embryo demise and a reduced rate of ongoing pregnancy, but the effect of bacteria on oocytes is unknown. This study examined the DNA within mouse oocytes after 12 hours’ incubation within human follicular fluids (n=5), which were collected from women undergoing in vitro fertilization (IVF) treatment. Each follicular fluid sample was cultured to detect the presence of bacteria. Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) was used to label DNA fragmentation in ovulated, non-fertilized mouse oocytes following in vitro incubation in human follicular fluid. The bacteria Streptococcus anginosus and Peptoniphilus spp., Lactobacillus gasseri (low-dose), L. gasseri (high-dose), Enterococcus faecalis, or Propionibacterium acnes were detected within the follicular fluids. The most severe DNA fragmentation was observed in oocytes incubated in the follicular fluids containing P. acnes or L. gasseri (high-dose). No DNA fragmentation was observed in the mouse oocytes incubated in the follicular fluid containing low-dose L. gasseri or E. faecalis. Low human oocyte fertilization rates (<29%) were associated with extensive fragmentation in mouse oocytes (80–100%). Bacteria colonizing human follicular fluid in vivo may cause DNA fragmentation in mouse oocytes following 12h of in vitro incubation. Follicular fluid bacteria may result in poor quality oocytes and/or embryos, leading to poor IVF outcomes.

CD9 Expression by Human Granulosa Cells and Platelets as a Predictor of Fertilization Success during IVF

Objective. To determine whether CD9 expression on human granulosa cells (GCs) and platelets could predict the success of conventional fertilization of human oocytes during in vitro fertilization (IVF). Methods. Thirty women undergoing IVF for nonmale factor infertility participated. Platelets from venous blood and GCs separated from retrieved oocytes were prepared for immunofluorescence. Flow cytometry quantified the percent of GCs expressing CD9, and CD9 surface density on GCs and platelets. Fertilization rate was determined for the total number of oocytes, and the number of mature oocytes per patient. Correlations tested for significant relationships (P < .05) between fertilization rates and CD9 expression. Results. CD9 surface density on human GCs is inversely correlated with fertilization rate of oocytes (P = .04), but the relationship was weak. Conclusion. More studies are needed to determine if CD9 expression on GCs would be useful for predicting conventional fertilization success during IVF.

Reducing CYP51 inhibits follicle-stimulating hormone induced resumption of mouse oocyte meiosis in vitro

Meiosis activating sterol, produced directly by lanosterol 14-alpha-demethylase (CYP51) during cholesterol biosynthesis, has been shown to promote the initiation of oocyte meiosis. However, the physiological significance of CYP51 action on oocyte meiosis in response to gonadotrophins' induction remained to be further explored. Herein, we analyzed the role of CYP51 in gonadotrophin-induced in vitro oocyte maturation via RNA interference (RNAi). We showed that although both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) significantly induced meiotic resumption in follicle-enclosed oocytes (FEOs), the effect of LH on oocyte meiosis resumption in FEOs was weaker than FSH. Moreover, both FSH and LH were able to upregulate CYP51 expression in cultured follicular granulosa cells when examined at 8 h or 12 h posttreatments, respectively. Interestingly, whereas knockdown of CYP51 expression via small interference RNA (siRNA) moderately blocked (23% reduction at 24 h) FSH-induced oocyte maturation [43% germinal vesicle breakdown (GVBD) rate in RNAi vs. 66% in control, P < 0.05] in FEOs, similar treatments showed no apparent effects on LH-induced FEO meiotic maturation (58% GVBD rate in RNAi vs. 63% in control, P > 0.05). Moreover, the results in a cumulus-enclosed oocytes (CEOs) model showed that approximately 30% of FSH-induced CEOs' meiotic resumption was blocked upon CYP51 knockdown by siRNAs. These findings suggest that FSH, partially at least, employs CYP51, and therefore the MAS pathway, to initiate oocyte meiosis.

The diagnostic value of seminal adenosine triphosphate (ATP) in an in vitro fertilization (IVF) program: Der diagnostische Wert von ATP in einem IVF-Programm

The level of adenosine triphosphate (ATP) was quantitated in semen samples used for in vitro fertilization of human oocytes. Seminal ATP level correlated with the concentration and percentage motility of spermatozoa but not with the in vitro fertilization rate of human oocytes. Seminal ATP measurement appears to have little diagnostic value in predicting the fertilizing capacity of spermatozoa as evaluated by the multivariate stepwise discriminant analysis.

Adhesion molecules and matrix proteins on human spermatozoa.

Ejaculated spermatozoa and spermatogenic cells express alpha- and beta-chains of beta 1, 3 and 4 integrins as well as their ligands fibronectin and laminin in an extended intra- and interindividual variation and in different patterns of location. The mRNA transcripts of these molecules were detectable by nested polymerase chain reaction in the spermatozoa. The conclusion of a functional competence of these adhesion molecules (AM) was supported by their relation to the results of the zona-free hamster oocyte penetration (HOP) test, the in vitro fertilization of human oocytes and cell attachment assays. AM labelling was influenced by the disintegration of the sperm plasma membrane, especially in seminal plasma, by progesterone, human follicular fluid and microorganisms, but was barely modified by sperm cryopreservation. Despite substantial advances in the knowledge about sperm adhesion molecules, many questions remain to be answered.

Human sperm movement assessed with the Hamilton-Thorn motility analyzer and in vitro fertilization.

The relationship between sperm movement characteristics obtained by computerized analysis and the in vitro fertilization rates of human oocytes was studied. In 144 consecutive in vitro fertilization treatments a sample of prepared semen was analysed by a Hamilton-Thorn Motility Analyzer. In addition a visual estimation of sperm count and motility was made. Significant correlations with the fertilization rate were found for all visual parameters. Of the computerized measurements, the mean velocities of motile spermatozoa and the concentration of motile cells were significantly correlated. The average path velocity correlated best (r = 0.42, P < 0.001). There was no relationship between the percentage of motile sperm showing hyperactivated movement and the fertilization rate. A forward stepwise logistic regression analysis selected the following variables of predictive value for fertilization: average path velocity, male factor infertility as indication for in vitro fertilization, motility and concentration, as measured by the Hamilton-Thorn analyzer. A logistic regression model to predict the cases with low (< 0.2) or high fertilization rates, included the average path velocity as a significant variable and classified the samples with 90% overall accuracy. movement characteristics of spermatozoa in culture medium, especially the average path velocity are of prognostic value in prediction of human oocyte fertilization rates.

2 year follow-up of children born after the transfer of embryos obtained by in vitro fertilization of human oocytes cryopreserved in low sodium medium

OBJECTIVE: To evaluate the growth and development of children born after the transfer of embryos obtained by in vitro fertilization of human oocytes cryopreserved in a choline-based freezing medium. DESIGN: Long term follow-up study in a medical institution. MATERIALS AND METHODS: Follow-up of 4 ICSI-singletons born between January 2000 and January 2004 in Halitus Medical Institute. Obstetric, peri- and post-natal data were collected and children were examined at 6, 12 and 24 months after birth. The four patients included in the study cryopreserved their oocytes (41 oocytes in total) between 1999 and 2003. Our oocyte cryopreservation program started in 1998. At the moment, 48 patients maintain 629 oocytes cryopreserved (13,1 oocytes/ patient); in 5 cases fertility preservation was the indication for oocyte cryopreservation. RESULTS: Four women, aged 30 ± 0.81(mean ± SD) gave birth to 4 singleton infants. Mean duration of female infertility was 5, 5 years (range 3 to 8 years). Patients had their 41 oocytes thawed; survival rate was: 41,4 % (17 oocytes), fertilization rate was 64,7% (11). Number of embryos transferred/ patient: 2, 4, 3 and 2 (2,75 ± 0,96). All transfers were performed on day 2. Mean birthweight of infants was 2870,75 ± 631 g (range: 2050 to 3550 g). There was 1 preterm delivery at 34 weeks of gestation; neonatal intensive care was required in this case. In all, 1 was a vaginal delivery and the other 3 were caesarean sections. Three healthy girls (75%) and one healthy baby boy (25%) were born. No perinatal mortality occurred. No post-partum complications were observed. At 6, 12 and 24 months of age, weights and heights of children were within normal parameters: mean weights: 7525, 10135 and 12562 g; and mean heights 63, 74,6 and 86,5 cm respectively. Neurological outcome of the four children at the age of 12 months showed normal development. CONCLUSIONS: The obstetric, peri- and post-natal outcome of the four babies was good. Further development of children was normal after 2 years of follow up. To our knowledge, this is the first report to present preliminary follow-up data of children born after oocyte cryopreservation in a low sodium medium. Careful evaluation is essential since choline based freezing technique is relatively new. These promising findings enhance the need for long-term follow-up of children born after this procedure.

Proteasome dynamics during maturation and fertilization of human oocytes

Proteasomes has been involved in the inactivation of the MPF (Metaphase Promoting Factor) and therefore in the progression from MI (Metaphase I) to MII (Metaphase II). Proteasomal inhibitors block fertilization. To better understand the function of proteasomes during oocyte meiosis and fertilization, we analyzed the distribution of proteasomes and poly-ubiquitinated proteins during both events after incubation with proteasome inhibitors MG132 and Velcade™. Descriptive and functional basic study. Human oocytes at germinal vesicle stage (GVs) were incubated during 17 h in maturation media supplemented with 10 mM MG132 (n = 57) or 100 nM Velcade™ (n = 61) at 37°C and 5% of CO2. To study the effect of proteasome inhibition during fertilization, a total of 29 supernumerary discarded normal fertilized oocytes (2 pronuclei, 2PNs) were incubated in the presence of the mentioned inhibitors. Antibodies against ab proteasomes subunits and FK1 (polyubiquitins proteins) were used for immunofluorescent labeling. Images were acquired by Confocal Microscopy and edited with Adobe Photoshop 7.0. Proteasome inhibition during in vitro maturation, arrested oocytes at different stages (MG132 and Velcade™ respectively): 14.0% and 16.4% at GV, 84.2% and 82.0% at GVBD, and 1.8% and 1.6% at MII (45% and 61% controls, (P<0.001)). The majority of GVBDs after MG132 were arrested at prometaphase stage after MG132 and at MI after Velcade™. Accumulations of poly-ubiquitin proteins in the cytoplasm were observed in all cases. After incubation of 2PNs with MG132 and Velcade™, 100% and 42.9% of them arrested at the syngamy stage respectively. In all cases, zygotes arrested with proteasome inhibitors show poly-ubiquitin proteins distributed in the cytoplasm and close to the nucleoli inside 2PNs. As expected, proteasomes were concentrated inside the 2PNs and closed to both PNs. Our results indicate that inhibition of proteasomes arrests cell cycle preventing in vitro maturation of human oocytes. This demonstrates the importance of proteasomes during the organization of the first metaphase plate suggesting a new role of the ubiquitin-proteasomes pathway in mammalian fertilization. Our data shows that proteasomal activity is essential for the decrease in MPF activity and completion of the first meiotic division in humans.

Follicle retinol binding protein levels accurately predict human oocyte fertilization potential

Molecular basis of human oocyte maturity and fertilization potential remains unclear. Proteins in human follicular fluid (HFF) are produced by follicular structure and provided from the transfer of blood plasma constituents. The aim of this study was to determine protein indicators for the oocytes maturation.

Citation classics in Fertility and Sterility, 1975–2004

Citation classics in Fertility and Sterility, 1975–2004

Escaping the tyranny of the embryo? A new approach to ART regulation based on UK and Australian experiences

The early legislative responses to fertilization of human oocytes in vitro exaggerated the protection of the embryo at the expense of the interests of other parties. Although more recent legislative changes have lessened this embryonic grip, it nonetheless still distorts legal thinking and is fundamentally in conflict with biological understanding. Drawing largely on experiences in the UK and Australia, a proposal is explored that reframes the legislative approach to the regulation of assisted reproductive technology (ART) with the following objectives: (i) to align more closely the legal and biological understandings of the earliest stages of human development; (ii) to place the legislative focus on objective, intent and outcome; and in the process (iii) to disentangle legally and conceptually the status of the embryo from that of the potential child. Experiences in the UK and Australia are drawn on, because these two jurisdictions have a common legal heritage and were among the earliest players both scientifically and legally but have pursued very different legal routes to their current legislative practices.

Deleted Mitochondrial DNA in Human Luteinized Granulosa Cells

The rearrangement of mitochondrial DNA in luteinized granulosa cells was determined in order to evaluate the fertilization capacity of oocytes and the development of embryos. Multiple deletions of mtDNA were found in luteinized granulosa cells from in vitro fertilization (IVF) patients. The 4977-base pair (bp) deletion was the most frequent deletion found in human granulosa cells. No significant difference was noted between mtDNA deletions of granulosa cells based on the fertilization capacity of oocytes and the development of embryos. To determine the relationship of proportions of mtDNA rearrangements with the aging process, granulosa cells were grouped into three different cohorts according to maternal age: younger than 32 years, between 32 and 37 years, and older than 37 years. No statistical correlation was noted between patient age and the frequency of occurrence of multiple mtDNA deletions. However, an increase in granulosa cell apoptosis was associated with an increase in mtDNA deletions. Accumulation of mtDNA deletions may contribute to mitochondrial dysfunction and impaired ATP production. We concluded that the accumulation of rearranged mtDNA in granulosa cells might not interfere with fertilization of human oocytes and further embryonic development; it was, however, associated with apoptosis processes.

In vitro maturation and in vitro fertilization of human oocytes.

AbstractIn vitro maturation (IVM) of human oocytes is an attractive technique for reducing the costs and averting the side effects of gonadotropin stimulation for in vitro fertilization (IVF). Edwards reported that human follicle oocytes could be matured in vitro when they were isolated from follicles and cultured in an appropriate system (1). The first successful fertilization of human oocytes matured in vitro was reported in 1969 (2). However, such a fertilized oocyte was not transferred back to patients until live birth of IVF of in vivo matured oocytes was achieved (3). A few IVF clinics have attempted to transfer embryos resulting from in vitro matured oocytes and have succeeded in getting the patient pregnant (4–13). However, only recently did IVM become clinically viable for treating infertile women in some IVF clinics (8,10–12). When compared with in vivo matured oocytes, the success rate is still low. The quality of oocytes after IVM appears to be suboptimal because embryos resulting from IVM show more frequent developmental block and transfer of such embryos results in low implantation and clinical pregnancy rates. Recent research advances, especially in the patient preparation, small follicle aspiration, and the oocyte culture system make it possible to adopt this new assisted reproductive technologies (ART) in IVF clinics. Women with infertility who have polycystic ovaries or polycystic ovarian syndrome (PCOS) should be considered for IVM treatment.KeywordsFollicular FluidCumulus CellPCOS PatientClinical Pregnancy RateHuman OocyteThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Efficiency of Percutaneous Testicular Sperm Aspiration as a Mode of Sperm Collection for Intracytoplasmic Sperm Injection in Nonobstructive Azoospermia

Efficiency of Percutaneous Testicular Sperm Aspiration as a Mode of Sperm Collection for Intracytoplasmic Sperm Injection in Nonobstructive Azoospermia

Sperm capacitation index (SCI) predicts pregnancy outcome with controlled ovarian hyperstimulation (COH) + intrauterine insemination (IUI)

Objective: The Sperm Penetration Assay (SPA) evaluates aspects of the ability of human sperm to complete certain processes necessary to achieve fertilization using zona-free hamster oocytes. There is controversy regarding the correlation between the SPA and fertilization of human oocytes or pregnancy outcome. An abnormal SPA is a good predictor of IVF + ICSI pregnancy outcome, but does not affect fertilization rates (Merryman, et al, ASRM abstract #P313, Orlando, FL, 2001). The SCI is the quantitative measurement of the SPA (average number of sperm penetrations per oocyte). Our objective was to determine the SCI score predictive of COH + IUI outcome. Clinical pregnancy per treatment cycle used as the outcome measurement.