Chromosome investigation in in vitro fertilization failure

Purpose A chromosomal complement of 227 human oocytes was studied to provide information on the frequency and type of chromosomal abnormalities in oocytes failing in vitro fertilization.Results Normal haploid chromosome complement was found in 54.6%; chromosomal abnormalities consisting of diploid sets were identified in 16.7% and aneuploidy was observed in 26%. Premature condensation of sperm chromosomes of the G1-phase was observed in 22.9% oocytes. Male infertility was correlated with an increase in the rate of aneuploidy when compared with tubal infertility. The rate of chromosome abnormalities for the oocytes recovered from women who had no fertilized oocytes was significantly higher compared to those with at least one oocyte fertilized.Conclusion A high frequency of chromosome abnormalities in unfertilized oocytes suggests that natural selection against chromosome abnormalities may occur even prior to fertilization.     

Sperm electron microscopy for the evaluation of in vitro fertilization failures

When performing IVF, the clinician is frequently confronted with the failure of fertilization. When the standard parameters to evaluate the male factor are "within normal limits," the conclusion is often made that the lack of fertilization is most likely due to "poor egg quality." These two cases demonstrate the fallacy of this approach and support a more rigorous evaluation of the male factor. Ultrastructural analysis of sperm is underutilized and, as demonstrated by these two cases, can play an essential role in this evaluation process.     

Cytologic screening and human papilloma virus test in women undergoing artificial fertilization

BACKGROUND: Infertile women in Sweden are offered in vitro fertilization (IVF) within the frame of the social security system. The present investigation was undertaken to evaluate the prevalence of genital human papilloma virus (HPV) infection in relation to the results of cytologic screening and to the infertility in these women. MATERIAL AND METHODS: Two hundred and fourteen women, mean age 32 years (range 20-40), admitted to the Center for Reproduction at Uppsala University Hospital for investigation of infertility and IVF were studied. Human papilloma virus tests were performed by a sensitive polymerase chain reaction-based technique in cervical smears obtained at a medical examination or during oocyte retrieval. Cytologic screening results were obtained from medical records or at the time of investigation. The infertile women were compared with 197 healthy female controls. RESULTS: Infertility resulted from female factors in 47% and male factors in 29% of the cases, and remained unexplained in 24%. Seven percent of the infertile women were HPV-positive compared with 9.1% of the controls. Only genital and oncogenic HPV types were identified. Human papilloma virus type 16 was most prevalent, and examination of the HPV 16 E6 gene showed that this prototype predominated over variants. No correlation was found between HPV infection and cause of infertility. Abnormal cytology was observed in 2.3% of the infertile women and 4.1% of the controls. CONCLUSIONS: Human papilloma virus infections might appear somewhat less frequently in infertile women admitted for IVF than in a control population. In both groups HPV infection was more common than cytologic abnormalities, possibly indicating that present HPV tests are more sensitive in detecting HPV infections than cytologic screening.     

Simultaneous analysis of cytoskeletal patterns and chromosome positioning in human fertilization failures

OBJECTIVE: To sequentially and reliably apply both tubulin immunocytochemistry (ICC) and fluorescence in situ hybridization (FISH) to human fertilization failures, thus providing a tool for a multiple analysis of arrest. DESIGN: Analysis of human fertilization failures at several stages of arrest. SETTING: Academic and clinical institutions. PATIENT(S): Consenting patients undergoing assisted reproduction technologies. INTERVENTION(S): Failed fertilizations displaying signs of activation without pronuclear development, or with the absence of polar body emission or cleavage 48 hours after insemination or microinjection were analyzed. Fertilization failures were fixed and processed for ICC. After data was collected the same samples were then subjected to FISH analysis using probes for chromosomes X, Y, and 18. MAIN OUTCOME MEASURE(S): Simultaneous ICC and FISH data on the same sample. RESULT(S): Sequential application of straightforward standard ICC and FISH techniques was not possible, as the morphologic features had been altered, microtubular patterns were not preserved, and many samples were rendered opaque. Only chromatin at the cell surface or outside the oocyte/zygote, such as metaphase II spindles or polar body nuclei, could be routinely probed for FISH after ICC. However, an increase in detergent-induced sample permeabilization as well as the removal of several steps usually performed for FISH made it possible to directly compare microtubular patterns and chromosome position, regardless of chromatin status. CONCLUSION(S): Analysis of specific proteins by immunocytochemistry and of chromosome status/positioning by FISH can be carried out sequentially in human fertilization failures, irrespective of the stage of arrest.     

Gamete quality in human in vitro fertilization

The success of in vitro fertilization and embryo transfer depends mainly on oocyte and sperm quality. As far as the oocyte is concerned, its fertilizability and capacity for subsequent development are related to intrinsic factors: quality and maturity, and extrinsic factors; quality of superovulation monitoring. These parameters have been widely studied using morphological (appearance of the oocyte-cumulus complex) and endocrine (plasma and follicular hormonology) criteria.     

Quality control in human in vitro fertilization

The implementation of suitable quality control (QC) is not only required for the accreditation of a human in vitro fertilization (IVF) laboratory, but is also fundamental to its success. Several assays have been employed to screen culture media and contact supplies. The suitability of one assay in particular, the mouse embryo assay (MEA), has been questioned over the years. Here we discuss how the conditions of such an assay, together with the stage of embryonic development used, have a profound effect on the outcome of the assay. Furthermore, by assessing embryos at multiple time points during the preimplantation period (rather than simply determining blastocyst formation), together with quantitating key parameters such as blastocyst cell number, it is possible to identify suboptimal components of a culture system. As well as identifying those components that result in outright embryonic demise, under the appropriate conditions the MEA can detect components that lead to impaired development. It is proposed that under the appropriate conditions, the MEA is a useful adjunct to quality control in human IVF, but several assays used in concert are better than a single test.     

Seasonality and human in vitro fertilization outcome

Seasonal changes in spontaneous fecundity have been observed in several human populations, but it is not clear whether the same applies to human in vitro fertilization–embryo transfer (IVF) procedures. In the present study, 2067 patients undergoing their first IVF attempt between the years 1998 and 2003 were grouped into four 'seasons' (December–February, March–May, June–August, September–November) according to the day on which they were administered human chorionic gonadotropin. Several parameters known to affect IVF outcome (including ovarian response to gonadotropins, sperm and oocyte quality, fertilization rate, embryo quality, pregnancy and implantation rates) were considered and potential changes among the four seasonal periods were analyzed. Moreover, some confounding variables (sperm quality, age, duration of infertility, indications for IVF) were controlled for. Overall, pregnancy rate per oocyte pick-up and per embryo transfer of 32.5% and 35.8%, respectively, as well as implantation rate of 18.9%, were recorded. None of the observed IVF-related parameters showed any significant change clearly related to a specific seasonal period, their fluctuations throughout the year being randomly determined. Ovarian responsiveness to gonadotropins, quality of gametes and embryos, and fertilization and implantation processes were apparently not significantly affected by seasonality. It is concluded that the results of a goodquality IVF program in humans are not significantly affected by the season of the year in which the IVF attempt is accomplished; therefore, season is not a relevant factor to be considered when planning an IVF treatment.     

Sperm antibodies and human in vitro fertilization

In order to directly evaluate the effects of sperm antibodies in human in vitro fertilization (IVF), the authors preincubated donor sperm in female sera containing sperm antibodies and then inseminated supernumerary human oocytes from a gamete intrafallopian transfer (GIFT) program. The sperm were incubated for 30 minutes in medium containing 20% serum with antisperm activity (Test); or no antisperm activity (Control) as assessed by the immunobead test (IBT). Each oocyte was inseminated with 1 to 2 X 10(5)/ml of the preincubated motile sperm with Control or Test treatments allocated on a random basis. Six positive sera were tested in 17 experiments, resulting in a fertilization rate of 41% (25/61) versus 84% (36/43) for controls (P less than 0.001). When considered individually, three of six positive sera caused significant inhibition. The only serum that gave complete inhibition had the highest titer for IgG (10,000) and lower IgA (100). Absorption with protein A reduced the IgG titer to less than 10 and removed the fertilization inhibitory activity. These results confirm that sperm antibodies from female sera can inhibit human IVF.     

Toxicity testing for human in vitro fertilization programs

Using a mouse embryo culture system, several procedures and materials associated with human in vitro fertilization protocols were tested for potential toxicity. Also, quality-control assays were performed for media prepared by nine different human in vitro fertilization programs. Detrimental effects upon embryo development were observed when culture media were exposed to the following substances: surgical instruments sterilized with Cidex or Cidex-7 or sterilized with ethylene oxide after packaging in Nest Protector Packs, various brands of surgical gloves, and various synthetic materials being evaluated as possible needle or catheter liners. Results from comparative testing of media and serum supplements prepared by different in vitro programs indicated a wide range in culture medium quality, as assayed by the ability of the reagents to support mouse embryo development. The importance of an animal model system available to all human in vitro programs for routine quality-control analysis and testing of novel uses of materials and innovative methods is discussed.     

Polyovular follicles associated with human in vitro fertilization

Polyovular follicles were found in 61 (24%) of 251 laparoscopies performed for in vitro fertilization. Of 898 follicles that contained at least one oocyte, 76 (8%) were polyovular. When the oocytes from polyovular follicles were assessed by morphologic criteria, it was found that 46 of the follicles contained oocytes that were discordant in maturity. Thus, oocytes can develop at different rates even though they are exposed to the same follicular fluid. It is possible that the microenvironment provided by the cellular investments of oocytes may correlate more precisely with oocyte maturity.     

Seasonality and human in vitro fertilization outcome.

Seasonal changes in spontaneous fecundity have been observed in several human populations, but it is not clear whether the same applies to human in vitro fertilization-embryo transfer (IVF) procedures. In the present study, 2067 patients undergoing their first IVF attempt between the years 1998 and 2003 were grouped into four 'seasons' (December-February, March-May, June-August, September-November) according to the day on which they were administered human chorionic gonadotropin. Several parameters known to affect IVF outcome (including ovarian response to gonadotropins, sperm and oocyte quality, fertilization rate, embryo quality, pregnancy and implantation rates) were considered and potential changes among the four seasonal periods were analyzed. Moreover, some confounding variables (sperm quality, age, duration of infertility, indications for IVF) were controlled for. Overall, pregnancy rate per oocyte pick-up and per embryo transfer of 32.5% and 35.8%, respectively, as well as implantation rate of 18.9%, were recorded. None of the observed IVF-related parameters showed any significant change clearly related to a specific seasonal period, their fluctuations throughout the year being randomly determined. Ovarian responsiveness to gonadotropins, quality of gametes and embryos, and fertilization and implantation processes were apparently not significantly affected by seasonality. It is concluded that the results of a good-quality IVF program in humans are not significantly affected by the season of the year in which the IVF attempt is accomplished; therefore, season is not a relevant factor to be considered when planning an IVF treatment.     

Endotoxins in culture medium for human in vitro fertilization

Endotoxins were detected in a few batches of culture medium during the authors' human in vitro fertilization program. Two distinct levels of endotoxins were assayed: greater than 1 ng/ml and less than 1 ng/ml. The source of endotoxin was traced to culture media obtained from a reputable manufacturing company. The incidence of fertilization per patient was not significantly affected by the presence of endotoxins, but fertilization assessed on the overall number of oocytes was significantly reduced (53%) when endotoxin levels were greater than 1 ng/ml compared with an assay negative for endotoxins (66%) (P = 0.047). The percentage of oocytes cleaving after the observation of two pronuclei was not significantly different, but the degree of fragments observed in the conceptus was significantly more severe if the endotoxin level reached 1 ng/ml. In this investigation, the incidence of pregnancy was 8% when the endotoxin level was greater than 1 ng/ml, 30% if less than 1 ng/ml, and 32% if no endotoxins were detected. This study suggests that, although endotoxins may be present in the culture medium at a deleterious level of at least 1 ng/ml, fertilization and cleavage will be obtained, but there will be a significant increase in the incidence of conceptuses with cytoplasmic fragments; this may result in a reduction in the incidence of pregnancy.     

Endotoxin-polluted medium in a human in vitro fertilization program

Optimal quality control measures to obtain the highest possible success rate for an in vitro fertilization and embryo transfer program is important in any program. Poor results obtained during a cycle where no pregnancies were achieved necessitated an investigation into possible causes. A suspected contamination of the multidistilled water utilized in medium preparation was confirmed. During a successful cycle with no endotoxins present, a pregnancy rate of 27.2% was achieved. This is higher than the 6.3% pregnancy rate achieved in another cycle where endotoxins were isolated in the media. This article illustrates cell morphologic and ultrastructural changes which could be ascribed to the presence of these endotoxins.     

Follicular size and number in human in vitro fertilization

In an attempt to maximize the success of in vitro fertilization (IVF) and embryo transfer (ET) as a treatment for human infertility, we have examined the relationship of follicular size and number to the rates of oocyte recovery, fertilization, cleavage, and ET in clomiphene citrate-stimulated cycles. The recovery of oocytes from follicles less than 20 mm in diameter was significantly reduced over that from larger follicles, and those oocytes that were obtained from smaller follicles showed a significantly lower rate of fertilization and cleavage. In addition, the overall chance that a patient would undergo ET was greater in a cycle in which more than one follicle 20 mm or larger was developing than in a cycle in which a single large follicle was developing. This latter observation suggests that attempts at laparoscopic oocyte retrieval should be confined to cycles in which more than one accessible large follicle is developing, thereby maximizing the success rate while minimizing the risk and expense for the patient.