Operative culdolaparoscopy: a new approach combining operative culdoscopy and minilaparoscopy.

The tendency is to use small cannulas for operative laparoscopy; however, working with these cannulas may have technical limitations. We developed a technique for performing appendectomy combining culdoscopy and minilaparoscopy. It uses 3- or 5-mm abdominal cannulas, and the large 10- or 12-mm cannula is inserted into the posterior vaginal fornix under laparoscopic surveillance. The vaginal port is used to introduce operative instruments and extract specimens, and for vision. Culdolaparoscopy avoids additional or large abdominal ports, thus overcoming limitations of small cannulas.

Laparoscopic leash: a simple technique to prevent specimen loss during operative laparoscopy.

BACKGROUND: Surgical specimens can be lost in the peritoneal cavity during operative laparoscopy. Although specimens left might cause no complications, peritonitis and adhesion formation have been reported, requiring subsequent laparoscopy or laparotomy. We report a simple technique to prevent loss of surgical specimens during laparoscopy. TECHNIQUE: A suture is placed through the specimen, and the trocar sleeve is removed. Free ends of the suture are held with a clamp outside the abdomen while the port is reinserted into the abdomen. The suture is pulled to see the specimen when necessary. When morcellation is required, the leashed area of the specimen is the last to be extracted. This procedure takes less than 2 minutes. EXPERIENCE: We have used this technique for longer than 1 year for 18 myomectomies and seven bilateral salpingo-oophorectomies. No specimens were lost in the peritoneal cavity, and there were no complications related to the procedure. CONCLUSION: The laparoscopic leash is a simple and reproducible preventive technique that adds insignificant time to operations but saves much time that might be wasted localizing a misplaced specimen.

Sperm integrity is critical for normal mitotic division and early embryonic development.

The human zygote relies on the paternal gamete to provide the centrosome component essential for the first mitotic division. It is not known whether normal centrosome function requires an intact spermatozoon, or whether donation of an isolated paternal centrosome component can result in normal zygotes and embryos. To explore this possibility, mature human oocytes were microinjected with either intact or dissected spermatozoa. Fertilization and cleavage rates were documented; nuclear and cytoskeletal changes were observed with fluorescent immunocytochemistry; and chromosomal normality was assessed with fluorescent in-situ hybridization. A pilot study was performed to identify cytoskeletal features suggestive of centrosome function. Unfertilized oocytes and tripronucleate (3PN) zygotes from in-vitro fertilization or intracytoplasmic sperm injection were assessed to confirm the sequence of the landmarks of human fertilization. Oocytes injected with mechanically-dissected spermatozoa appear to be capable of normal pronuclear formation and embryonic cleavage, but do not undergo normal mitotic division. Although decondensed, apposed nuclei are noted in combination with diffuse cytoskeleton assembly, no spindle was detected in any zygote resulting from the injection of a dissected spermatozoon. Analysis of selected embryos resulting from dissected sperm injection revealed chromosomal mosaicism in the majority of specimens. The lack of a bipolar spindle, in combination with chromosomal mosaicism, suggests abnormalities of the mitotic apparatus when sperm integrity is impaired following dissection.

The role of structural integrity of the fertilising spermatozoon in early human embryogenesis.

While the fertilising spermatozoon supplies the active centre directing the human zygote's first mitotic division, the relative contributions of the sperm head and tail (as well as the importance of the sperm's general structural integrity) to subsequent developmental processes remain incompletely studied. The sperm nucleus contains paternal chromatin necessary for restoration of a diploid genome, but the functional role of the sperm tail (either attached or dissected) in early human embryonic growth is not known. In this investigation using oocytes donated by in vitro fertilisation patients, human oocytes were injected with isolated sperm heads (n = 73), isolated sperm flagella (n = 11) or both (dissected sperm heads + free sperm tails, n = 26). The formation of bipronucleate zygotes was recorded for each method. Among oocytes surviving injection with isolated sperm heads, 44 of 66 (67%) formed two pronuclei. Of oocytes receiving only sperm tails, 2 of 11 (18%) displayed two pronuclei, but a single polar body was evident in both cases. When dissected spermatozoa parts (head + tail) were jointly injected, 12 of 26 (46%) developed two pronuclei. From embryos resulting from each of these three fertilisation regimes, blastomere biopsies were obtained and subjected to multiprobe fluorescent in situ hybridisation (FISH) analysis to detect mosaicism or aneuploidy arising from these experimental treatments. Only embryos with growth sufficient to permit sampling of at least two blastomeres were evaluated, and FISH analysis was successful in 25 of 29 (86%) embryos tested. Of 12 embryos derived from injection of an isolated sperm head, only one was normal diploid; the remaining 11 were mosaic. Both embryos resulting from injection of an unattached sperm tail were mosaic. Of 11 embryos generated from oocyte injection with sperm head + tail segments, 10 (91%) were mosaic and only one was normal diploid. Results from this study show that injection of isolated sperm segments can permit oocyte activation and bipronuclear formation. However, a high rate of mosaicism in human embryos originating from disrupted sperm or sperm components suggests that more than a 'sum of parts' is needed for later development. The structural integrity of the intact fertilising spermatozoon appears to contribute to normal human early embryogenesis.

Incidence of sperm aneuploidy in relation to semen characteristics and assisted reproductive outcome.

OBJECTIVE: To evaluate the incidence of sperm aneuploidy in men screened for infertility and identify any eventual relation with assisted reproductive outcome. DESIGN: Controlled prospective study. SETTING: University hospital-based IVF program. PATIENT(S): Infertile couples who were screened for sperm aneuploidy and evaluated for IVF treatment. INTERVENTION(S): Fluorescence in situ hybridization was used to identify chromosomes 18, 21, X, and Y. The assisted reproductive techniques of IVF and intracytoplasmic sperm injection were used for infertility treatment. MAIN OUTCOME MEASURE(S): The incidence of sperm aneuploidy, semen parameters, fertilization rate, pregnancy characteristics, and rate of neonatal malformations were determined. RESULT(S): Oligozoospermic and teratozoospermic men had a significantly higher incidence of chromosomal abnormalities than men with normal semen parameters (2.7% vs. 1.8%). The increased frequency of sperm aneuploidy did not appear to affect pregnancy losses or the occurrence of neonatal malformations. CONCLUSION(S): Suboptimal semen samples had a higher incidence of aneuploidy. In this study, the increased frequency of chromosomal abnormalities did not have a direct effect on the fertilization rate, pregnancy characteristics, or neonatal outcome.

Human glucosamine-6-phosphate isomerase, a homologue of hamster oscillin, does not appear to be involved in Ca2+ release in mammalian oocytes.

Injections of cytosolic preparations from mammalian sperm into oocytes have been shown to trigger calcium [Ca2+]i oscillations and initiate activation of development. Recently, a protein isolated from hamster sperm has been suggested to be involved in the generation of these oscillations and it was named "oscillin." The human homologue of hamster oscillin is glucosamine 6-phosphate isomerase (GPI, EC no. 5.3.1.10), an enzyme so far described to be involved in hexose phosphate metabolism. To assess the role of GPI on Ca2+ signaling, a human recombinant protein was generated in a prokaryotic system and injected into fura-2-dextran-loaded metaphase II (MII) mouse oocytes. Injection of recombinant GPI failed to induce Ca2+ responses in 12/12 injected MII oocytes despite the fact that the recombinant GPI was active as assessed by an enzymatic assay. Injection of buffer (0/6 oocytes) or fructose-6-phosphate, a product of GPI enzymatic reaction (0/5 oocytes), also failed to initiate Ca2+ responses. Conversely, injections of sperm cytosolic factor induced [Ca2+]i oscillations in all 17/17 oocytes. In addition, injection of recombinant GPI or GPI mRNA failed to induce parthenogenetic activation (0/30 oocytes). Immunofluorescence studies using an anti-GPI polyclonal antibody (GK) resulted in localization of GPI to the sperm's equatorial region. Incubation of the GK antibody with sperm extracts failed to block the [Ca2+]i responses induced by these extracts. Moreover, near complete depletion of GPI from sperm fractions by immunoprecipitation did not impair the ability of these fractions to induce [Ca2+]i oscillations. In summary, our results support the role of a sperm cytosolic component(s) in the generation of [Ca2+]i oscillations during mammalian fertilization, although a protein other than GPI/oscillin is likely to be the active calcium releasing factor.

A comparison of human spermatozoa immunolabeling features using xenogenic reagents for centrosomal proteins.

The centrosome is an organelle essential to proper chromosomal migration and normal cell growth. In the human, the centrosome is comprised of two centrioles and the pericentriolar cytosol; its control of embryo cleavage processes derives from its role as a locus for spindle organisation. At fertilisation, it is the human sperm centrosome that is responsible for ordering these processes, as the oocyte appears not to contain working centrosomal structures. Abnormalities in fertilisation or early embryo cleavage could be related to impaired sperm centrosome structure or function in some cases. While potential future treatments of infertility due to a defective centrosome could involve use of a donor centrosome to restore normal cell development, such an approach would depend on accurate localisation of this organelle for subsequent transplantation. To locate centrosomal components in the heads and tails of human spermatozoa, labeling was performed on intact spermatozoa using antibodies of known specificity to highly-conserved centrosomal elements. Following general mapping of immunofluorescent signals, unlabeled sperm were dissected to form head/tail sperm fragments which were then separately tested. Distribution of centrosomal proteins in head and tail fragments was assayed for each separation method. Three reagents were compared: 1) rabbit anti-mitotic spindle protein (anti-MSP) antibody, 2) rabbit polyclonal centriole-specific antibodies, and 3) mouse monoclonal anti-MPM-2 (a centrosome phospoprotein) antibody. Of these, anti-MPM-2 antibody appeared to be the most reliable, labeling centrosomal elements in 63% (n = 1,386) of treated spermatozoa. Sequential utilization of n-butylamine to effect head/tail separation followed by anti-MPM-2 antibody labeling was a satisfactory method of centrosome localisation. Microextraction of centrosomes and pericentriolar matrix identified by this method awaits further testing.

H-Y antigen expression patterns in human X- and Y-chromosome-bearing spermatozoa.

PROBLEM: Restricted expression of H-Y antigen on Y-chromosome-bearing sperm has been reported in some species, although such preferential expression for H-Y antigen in human sperm has yet to be described. In this study, an immunomagnetic approach was used to characterize antigen expression patterns as a function of sex-chromosome content. METHOD OF STUDY: Human sperm was treated with monoclonal immunoglobulin (Ig) M antibodies directed against H-Y antigen. This preparation then was incubated with sheep antimouse IgM antibody affixed to paramagnetic beads, which then were exposed to a magnetic field and sorted. X- and Y-chromosome frequencies in the two subgroups of sperm were assayed by multiprobe fluorescent in situ hybridization (FISH). RESULTS: Sperm were immunomagnetically separated into two populations: a reactive group (presumably, H-Y Ag+); and a nonreactive group (presumably, H-Y Ag-). Triple-color FISH analysis of 1,600 spermatozoa (800 in each group) showed the antigen's expression to be somewhat more prevalent among Y-chromosome-bearing sperm (54.1%), but a large proportion of Y-chromosome-bearing sperm (49.0%) did not express this antigen. The difference was not significant (P = 0.43). CONCLUSIONS: The expression of H-Y antigen has a slightly higher frequency in human sperm containing the Y-chromosome, but its expression among X-chromosome-bearing sperm also is considerable. Current immunologic techniques relying on this antigen are unlikely to effect the sex selection of human sperm.

Effect of parental age on fertilization and pregnancy characteristics in couples treated by intracytoplasmic sperm injection.

The purpose of this study was to investigate any influence of maternal and/or paternal age on gamete characteristics and pregnancy outcomes in intracytoplasmic sperm injection (ICSI) cycles. In all, 821 consecutive ICSI cases were analysed retrospectively. While a significant linear decline in semen volume was detected, no significant differences in the concentration, motility or morphology of the spermatozoa were found with paternal ageing. A significant decline in the number of oocytes retrieved and the number of mature oocytes obtained was found with advancing maternal age. An increase in the occurrence of digyny was noted with parental ageing, while no difference in single or bipronuclear fertilization was found. Older women had a decreased incidence of single pronucleus formation and an increase in digyny, but no significant difference in the percentage of oocytes that underwent two-pronuclear fertilization was detected with regard to maternal ageing. Pregnancy outcomes were not influenced by the age of the male partner, while a strong negative correlation was found with maternal ageing. To better analyse male partner ageing as a factor affecting pregnancy outcome, we analysed a subgroup of patients with a female partner aged <35 years who underwent ICSI. No paternal influence on ICSI pregnancy outcome was found in this subgroup of patients. We conclude that the influence on pregnancy outcome after ICSI is related mostly to maternal and not paternal age.

Human sperm cytosolic factor triggers Ca2+ oscillations and overcomes activation failure of mammalian oocytes.

Among the possible mechanisms of oocyte activation after sperm penetration, it appears most likely that a protein released by the spermatozoon elicits a calcium elevation in the ooplasm. To further test this idea, cytosolic factors obtained from human spermatozoa by two different methods, freezing-thawing and sonication, were injected into mouse oocytes following which intracellular calcium release was measured. Of a total of 42 mouse oocytes, a pattern of calcium oscillations was observed in nine out of 16 oocytes injected with sonicated fraction, in all of eight oocytes with the frozen-thawed fraction and in none of 18 control oocytes. Injection of the frozen-thawed fraction also produced regular calcium oscillations in all of five in-vitro matured human oocytes. To assess the putative factor's ability to support fertilization, human oocytes that were not activated by prior intracytoplasmic injection of spermatozoa (ICSI) and round spermatids were reinjected with the frozen-thawed sperm fraction. Of 23 human oocytes which remained unfertilized after ICSI, 19 became activated after injection with sperm cytosolic factor; eight showed two pronuclei, three one pronucleus and eight showed three or more pronuclei. Of 11 oocytes unfertilized after prior round spermatid injection, two developed two pronuclei, four developed one pronucleus and two had three or more pronuclei. Cytogenetic analysis by fluorescence in-situ hybridization confirmed the existence of a male pronucleus in eight out of nine such zygotes displaying two or more pronuclei. Thus, human sperm extracts activated mouse and human oocytes after injection, as judged by calcium flux patterns in conjunction with male pronucleus formation.

The human sperm centrosome is responsible for normal syngamy and early embryonic development.

As early as 1887, it was postulated that the mature oocyte possesses all of the elements necessary for embryonic development with the exception of an active division centre, and that the spermatozoon contains such a centre, but lacks the substrate in which to operate. This division centre is called the centrosome. The precise definition of this structure is still a subject for debate. It consists of two centrioles in a perpendicular arrangement and pericentriolar material, and is considered to be responsible for nucleation of microtubules and the formation of the mitotic spindle. There is a paternal pattern of inheritance of the centrosome in humans; thus, human oocytes lack centrioles but the spermatozoa carry two. At gamete fusion the sperm tail is incorporated into the ooplasm, and the centriolar region forms the sperm aster while the sperm head is decondensing; this aster acts to guide the female pronucleus towards the male pronucleus. The centriole duplicates during the pronuclear stage, and at syngamy centrioles are found at opposite poles of the first cleavage. The centrosome has several implications for human infertility. It is possible that immotile or nonprogressively motile spermatozoa may possess centriolar abnormalities or an absence of centrioles. Similarly, antisperm antibodies against centrioles may be responsible for mitotic arrest. One way of solving this problem would be the use of donor centrosomes. To this end, we have assessed the ability of embryos injected with physically separated sperm segments (head only, head and tail separated or isolated tail) to develop normally. Fluorescent in situ hybridization revealed an almost universal mosaicism in these embryos, suggesting that physical disruption of the spermatozoa compromises the ability of the centrosome to function in the zygote. Thus far, centrosome donation with centriole-carrier flagellae obtained by this dissection method does not appear to be feasible.

Evolution of pregnancies and initial follow-up of newborns delivered after intracytoplasmic sperm injection.

OBJECTIVE: To analyze the in vivo development of embryos conceived after intracytoplasmic sperm injection (ICSI), as well as obstetric outcome, occurrence of chromosomal abnormalities, and rate of congenital malformations in neonates born as a result of this treatment. DESIGN: Retrospective study. SETTING: University-based in vitro fertilization (IVF) clinic. PATIENTS: A total of 751 couples in whom the male partners were presumed to be the cause of repeated failed IVF attempts or whose semen parameters were unacceptable for conventional IVF treatment. INTERVENTIONS: Analysis of pregnancies resulting from 987 ICSI cycles; pregnancy outcome data were obtained from the records of obstetrician-gynecologists and/or pediatricians. MAIN OUTCOME MEASURES: Pregnancy rates, obstetric outcome, and frequency of chromosomal abnormalities and congenital malformations. RESULTS: The overall clinical pregnancy (fetal heartbeat) rate was 44.3%, with a resultant delivery rate per ICSI cycle of 38.7% (n=382). In 8 of 11 miscarriages for which cytogenetic data were available, an autosomal trisomy was found, and 7 additional pregnancies were terminated because of a chromosomal abnormality after prenatal diagnosis. There was an equal distribution of vaginal vs cesarean deliveries (n=192 and n=190, respectively). Of the 578 neonates resulting from treatment by ICSI, 15 (2.6%) presented with congenital abnormalities (9 major and 6 minor abnormalities). However, this frequency of malformations is lower than that observed in offspring born after standard IVF at our institution. Furthermore, when pregnancy outcome of ICSI vs IVF was analyzed in terms of semen origin, no differences were found in the frequency of miscarriages or in the rate of congenital malformations. CONCLUSIONS: The evolution of pregnancies and occurrence of congenital malformations following treatment by ICSI were within the range observed with standard in vitro fertilization.

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 sperm immobilization prior to intracytoplasmic sperm injection (ICSI) influences fertilization by immature spermatozoa. Of the 837 ICSI cycles evaluated, 81 were performed with epididymal or testicular spermatozoa; 35 cycles with epididymal spermatozoa immobilized in the standard fashion resulted in fertilization and pregnancy rates of 48.3 and 51.4% respectively. When a more aggressive sperm immobilization technique (i.e. permanently crimping the sperm flagellum between the midpiece and the rest of the tail) was applied in 17 cycles, the resultant fertilization and pregnancy rates were significantly (P < 0.05) higher: 82.0 and 82.4% respectively. Similar increases in fertilization and ensuing pregnancy rates were also observed in ICSI cycles with the aggressive immobilization of frozen-thawed epididymal spermatozoa (eight cycles) versus standard immobilization (16 cycles). However, the fertilization rates for ICSI using testicular spermatozoa (five cycles) were basically the same, regardless of the immobilization technique. Furthermore, for ejaculated spermatozoa (756 cycles), the fertilization rates following aggressive sperm immobilization were also positively affected (73.4%), although no statistical differences in the clinical pregnancy rates were found. Because aggressive immobilization appears to affect sperm membrane permeabilization, the enhanced fertilization patterns observed in immature spermatozoa following aggressive immobilization may suggest a different membrane constitution in these spermatozoa. These findings indicate that immature gametes may require additional manipulation to enhance the post-ICSI events essential for adequate nuclear decondensation.

Oolemma characteristics in relation to survival and fertilization patterns of oocytes treated by intracytoplasmic sperm injection.

The aim of this study was to analyse the various reactions displayed by the oolemma to the penetrating pipette during intracytoplasmic sperm injection (ICSI) and correlate them with clinical factors, oocyte survival and fertilization patterns. Three types of oolemma responses were observed: normal breakage, when the injection needle created an invagination that ruptured at the approximate centre of the egg; sudden breakage, when the membrane broke without creating a funnel; and difficult breakage, when the membrane did not break or broke after several penetration attempts. A total of 2928 oocytes were analysed with the following observations: 73.9% (n = 2164) experienced normal breakage, 11.8% (n = 345) sudden breakage, and 14. 3% (n = 419) difficult breakage. The survival rate and number of normally fertilized oocytes were significantly lower and the incidence of digynic oocytes was significantly higher in the sudden breakage group; furthermore, in this group a significantly shorter length of stimulation was observed along with lower serum oestradiol concentrations when compared to oocytes experiencing normal and difficult breakage patterns. These recorded patterns were predictive of the survival and fertilization ability of the injected oocytes, as well as the incidence of digyny. The link between membrane behaviour and various clinical parameters appears to indicate a correlation between the modality of stimulation and oolemma characteristics.

Genetics of abnormal human fertilization.

The purpose of this study was to assess the genetic status of abnormal zygotes following assisted fertilization. Dispermic, monopronucleated and digynic zygotes were allowed to cleave intact or after enucleation, and on the biopsied blastomeres, multiplex polymerase chain reaction and fluorescent in-situ hybridization were performed. It was found that the distal pronucleus was usually male in origin in dispermic embryos, and that the sex ratio was restored when they were enucleated; however, they became mosaic at metaphase and their genetic heterogeneity was not restored after enucleation. Monopronucleated zygotes derived from standard in-vitro insemination can be transferred to the patient, since they usually showed normal diploid complement in their cells. On the contrary, single-pronucleated zygotes derived from intracytoplasmic sperm injection were usually activated parthenogenetically, but not fertilized. Digynic embryos, unlike dispermic ones, had a very low incidence of mosaicism, and when present, such mosaicism originated at a later embryo division. Most of the digynic embryos were triploid, indicating that the first division was normal and bipolar; moreover, when the female pronucleus was removed, they became diploid and their genetic status was considered normal. The recognition and understanding of fertilization abnormalities allow the identification of methods leading to their avoidance or correction.