Common technical errors in hysterosalpingography.

Hysterosalpingograms from 100 consecutive patients referred for in vitro fertilization were reviewed to evaluate the adequacy of visualization of the uterine cavity. In 17 cases the hysterosalpingogram failed to demonstrate the entire uterine cavity. The most common reason for failure was an axial view of the uterus secondary to inadequate traction on the cervix in 82% (14/17) of the cases, followed by obstructed visualization of the lower uterine cavity and endocervical canal by the delivery catheter bulb in 21% (3/17). A speculum left in the vagina obscured visualization of the endocervical canal in 21% (3/17) of the cases. There were no significant differences in the mean number of radiographic exposures between the adequate and inadequate groups (4.7 vs. 5.9). This study suggests that failing (1) to remove the speculum before injecting contrast, (2) to evaluate the lower uterus and endocervix when using an intrauterine catheter, or (3) to place adequate traction on the cervix, may result in inadequate visualization of the uterine cavity and a need to repeat the study.     

A preclinical evaluation of pronuclear formation by microinjection of human spermatozoa into human oocytes

In vitro fertilization (IVF) is recognized as an accepted treatment for male infertility. However, the fertilization rate is significantly lower than the fertilization rate of other IVF patient groups. Some male factor infertility patients still have a basic semen quality too poor for treatment by IVF. Microinjection of a spermatozoon directly into ooplasm has been recommended to assist fertilization in this subfertile population. This study found that oocytes from 5 of 11 patients microinjected with human spermatozoa demonstrated successful pronuclear formation and correlated with the incidence of pregnancy in these patients transferred with same-source oocytes inseminated by standard protocols. This initial evidence promotes the supposition of clinical feasibility of assisted fertilization by sperm microinjection.     

Does ICSI require acrosomal disruption? An ultrastructural study

BACKGROUND: Aggressive immobilization of sperm prior to ICSI significantly improves fertilization rates, but the mechanism of this effect is not yet clear. This study was performed in order to assess the characteristics of mechanically immobilized human sperm by transmission electron microscope (TEM). METHODS: Sperm obtained from ejaculated semen samples from three different donors were immobilized in a standard manner for ICSI. They were then injected into the perivitelline space of mouse oocytes in order to be able to locate them by TEM. Intact motile sperm injected subzonally served as controls (n=160). Finally, the 'carrier' oocytes were fixed and processed for TEM. RESULTS: A total of 300 sperm were mechanically immobilized and inserted into the perivitelline space of mouse oocytes. Ultrathin sections revealed consistent alterations in the acrosomal region including disruption of the plasma membrane, and disruption, vesiculation or even loss of the acrosome. Thus, all of the sperm assessed had undergone some disorganization of the head, in contrast to a majority of control sperm. CONCLUSIONS: Immobilization of sperm for ICSI by compressing and rolling the sperm tails induces a variable disruption and sometimes loss of the acrosome. This could well be a reason for the higher success rates when ICSI is performed using immobilized sperm.     

Aggressive sperm immobilization prior to intracytoplasmic sperm injection with immature spermatozoa improves fertilization and pregnancy rates

This study was conducted to determine whether the mode of spermimmobilization prior to intracytoplasmic sperm injection (ICSI)influences fertilization by immature spermatozoa. Of the 837ICSI cycles evaluated, 81 were performed with epididymal ortesticular spermatozoa; 35 cycles with epididymal spermatozoaimmobilized in the standard fashion resulted in fertilizationand pregnancy rates of 48.3 and 51.4% respectively. When a moreaggressive sperm immobilization technique (i.e. permanentlycrimping the sperm fiagellum between the midpiece and the restof the tail) was applied in 17 cycles, the resultant fertilizationand pregnancy rates were significantly (P < 0.05) higher:82.0 and 82.4% respectively. Similar increases in fertilizationand ensuing pregnancy rates were also observed in ICSI cycleswith the aggressive immobilization of frozen-thawed epididymalspermatozoa (eight cycles) versus standard immobilization (16cycles). However, the fertilization rates for ICSI using testicularspermatozoa (five cycles) were basically the same, regardlessof the immobilization technique. Furthermore, for ejaculatedspermatozoa (756 cycles), the fertilization rates followingaggressive sperm immobilization were also positively affected(73.4%), although no statistical differences in the clinicalpregnancy rates were found. Because aggressive immobilizationappears to affect sperm membrane pennea-bilization, the enhancedfertilization patterns observed in immature spermatozoa followingaggressive immobilization may suggest a different membrane constitutionin these spermatozoa. These findings indicate that immaturegametes may require additional manipulation to enhance the post-ICSIevents essential for adequate nuclear decon-densation.     

Heat shock protein expression during gametogenesis and embryogenesis

When cells are subjected to various stress factors, they increase the production of a group of proteins called heat shock proteins (hsp). Heat shock proteins are highly conserved proteins present in organisms ranging from bacteria to man. Heat shock proteins enable cells to survive adverse environmental conditions by preventing protein denaturation. Thus the physiological and pathological potential of hsps is enormous and has been studied widely over the past two decades. The presence or absence of hsps influences almost every aspect of reproduction. They are among the first proteins produced during mammalian embryo development. In this report, the production of hsps in gametogenesis and early embryo development is described. It has been suggested that prolonged and asymptomatic infections trigger immunity to microbial hsp epitopes that are also expressed in man. This may be relevant for human reproduction, since many couples with fertility problems have had a previous genital tract infection. Antibodies to bacterial and human hsps are present at high titers in sera of many patients undergoing in vitro fertilization. In a mouse embryo culture model, these antibodies impaired the mouse embryo development at unique developmental stages. The gross morphology of these embryos resembled cells undergoing apoptosis. The TUNEL (terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling) staining pattern, which is a common marker of apoptosis, revealed that embryos cultured in the presence of hsp antibodies stained TUNEL-positive more often than unexposed embryos. These data extend preexisting findings showing the detrimental effect of immune sensitization to hsps on embryo development.     

Autologous Endometrial Co-culture in Patients with Repeated Failures of Implantation After In Vitro Fertilization–Embryo Transfer

Purpose: Our purpose was to evaluate the effect of co-culture on preembryo development and clinical outcome. Methods: Enrolled patients underwent a luteal-phase endometrial biopsy. The tissue was then enzymatically digested (collagenase) and the stromal and glandular cells were separated by differential sedimentation rates. These cells were cultured to confluence, released, and then cryopreserved until the patient's in vitro fertilization (IVF)–embryo transfer (ET) cycle. All normally fertilized oocytes were then placed on the co-cultured cells until transfer on day 3. Preembryo development on co-culture was compared to that in the patient's noncocultured previous cycle. Implantation and clinical pregnancy rates were compared to those in a control group of patients undergoing IVF during the study period who were matched for age, stimulation protocol, number of oocytes retrieved, and preembryos transferred. Results: Twenty-nine women underwent 31 cycles of IVF-ET. On day 3 the overall mean number of blastomeres per preembryo on co-culture compared to that in the patient's previous cycle was 6.3 ± 1.8 vs. 5.6 ± 1.2 (P = 0.04). The average percentage of cytoplasmic fragments on co-culture compared to the previous cycle was 16 ± 9% vs. 19 ± 9% (P = 0.32). At transfer, after preembryo selection, the mean number of blastomeres per preembryo on co-culture compared to that in the patient's previous cycle was 6.8 ± 1.6 vs. 6.6 ± 1.3 (P = 0.5). The implantation and clinical pregnancy rates between co-culture and the matched control group were 15% (14/93) vs. 13% (16/124) (P = 0.79) and 29% (9/31) vs. 25% (10/40) (P = 0.45). Conclusions: There was a significant improvement in the average number of blastomeres per preembryo on co-culture compared to that in the patient's previous noncoculture cycle. The overall implantation and clinical pregnancy rates between co-culture and a matched control group were not significantly different.     

Preimplantation genetic diagnosis for retinoblastoma: the first reported liveborn

PURPOSE: To develop an accurate mutation analysis procedure for retinoblastoma gene (RB1) mutation, which is sensitive at the single-cell level, and to use in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) to achieve pregnancies without retinoblastoma. DESIGN: Case report. METHODS: Twelve day 3 embryos, obtained by IVF with intracytoplasmic sperm injection, underwent single-cell DNA testing via polymerase chain reaction and restriction enzyme analysis to detect the presence of a paternal RB1 mutation. Embryos were diagnosed as being unaffected and were transferred to the uterus on day 5. MAIN OUTCOME MEASURES: Achieving a healthy pregnancy and delivery, assessed by clinical presentation, fundus photography, and RB1 molecular analysis. RESULTS: A singleton pregnancy was achieved, and a child without retinoblastoma was born. The absence of the paternal RB1 mutation was confirmed on a sample of peripheral blood from the newborn. CONCLUSIONS: We are first to report a successful human liveborn, delivered after IVF with preimplantation genetic diagnosis for retinoblastoma. The successful result indicates that preimplantation genetic diagnosis exists for this genetic disease and may represent a viable alternative to prenatal diagnosis with the subsequent option of terminating an affected pregnancy.