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Morphology and histology of the male reproductive system in two species of internally inseminating South American catfishes, Trachelyopterus lucenai and T. galeatus (Teleostei: Auchenipteridae).

As part of an effort to characterize reproductive modifications in internally inseminating catfishes, ovaries and male reproductive systems were examined histologically in two species of auchenipterid catfishes, Trachelyopterus lucenai and T. galeatus, from southeastern Brazil. Internal insemination was documented in both species by the presence of sperm within ovaries. Although there is some variation in gross morphology of the male reproductive systems between the two species, both have four main regions: spermatogenic lobes, sperm storage regions, and secretory and storage regions of the seminal vesicle. In both species, the anterior portion of the reproductive system is spermatogenic and divided into numerous finger-like lobes. Posterior to the spermatogenic area is the storage region of the seminal vesicle, a large median structure with a honeycomb-like appearance. This region is consistently larger in T. lucenai. Attached to the storage region of the seminal vesicle in both species are secretory lobes comprised of tubules lined by secretory cells. These lobes in T. lucenai are small and located on the anterior aspect of the storage region of the seminal vesicle, whereas in T. galeatus the lobes are much larger and located laterally. The sperm storage regions of T. lucenai consist of two large lobes located ventral to the storage region of the seminal vesicle. Highly compact sperm packets (spermatozeugmata) fill the lumina of the ramifying tubules of these regions. Each spermatozeugma consists of elongate nuclei tightly arranged parallel to one another. In T. galeatus two distinct sperm storage regions are present. Just posterior to the spermatogenic lobes a series of small lobes serve as anterior sperm storage regions. Posterior to the secretory lobes of the seminal vesicle is a series of lobes, at the most posterior aspect of the reproductive tract, that serve as posterior sperm storage regions. Both are identical, histologically, to the sperm storage regions of T. lucenai. An absence of compact spermatozeugmata in the T. galeatus specimens may be related to variations in their sexual activity. The descriptions presented here allow for consistent terminology for comparison of regions of the male reproductive system based on presumed function.

Animals↗

Semen preparation techniques for intrauterine insemination.

BACKGROUND: Semen preparation techniques for assisted reproduction, including intrauterine insemination (IUI), were developed to separate the motile morphological normal spermatozoa. Leucocytes, bacteria and dead spermatozoa produce oxygen radicals that negatively influence the ability to fertilize the egg. The yield of as many motile, morphologically normal spermatozoa might influence treatment choices and therefore outcomes. OBJECTIVES: To compare the effectiveness of gradient, swim-up, or wash and centrifugation in subfertile couples undergoing intrauterine insemination (IUI) on clinical outcome as well as on semen parameters. SEARCH STRATEGY: We searched the Menstrual Disorders and Subfertility Group's trials register (30 June 2003), MEDLINE (1966 to July 2003), EMBASE (1980 to July 2003), Science Direct Database (1966 to July 2003), Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library Issue 3, 2003), National Research Register (2000 to Issue 2, 2003), Biological Abstracts (2000 to June 2003), CINAHL (1982 to July 2003) and reference lists of relevant articles. We also contacted experts and authors in the field. SELECTION CRITERIA: Parallel randomised controlled trials (RCTs), comparing the efficacy of semen preparation techniques used for subfertile couples undergoing IUI, were included. RCTs or split sample studies examining semen parameters after different semen preparation techniques were also included. DATA COLLECTION AND ANALYSIS: Two reviewers independently assessed trial quality and extracted data. Study authors were contacted for additional information. MAIN RESULTS: Two randomised controlled trials comparing clinical outcomes, including 81 participants in total, were included in the meta-analysis (Dodson 1998-I/ II; Xu 2000-I/ II). Both studies compared swim-up technique versus gradient technique in 65 subfertile couples undergoing IUI. One study compared the effectiveness of both techniques with wash technique. No trials reported the primary outcome of live birth. There was no statistically significant difference between pregnancy rates (PR) for swim-up versus gradient / wash centrifuge (Peto OR 0.55, 95% CI 0.17 to 1.76; Peto OR 1.74, 95% CI 0.2 to 14.9; PR/ couple swim-up 20%, gradient 40%, wash 12.5%) or gradient versus wash centrifuge (Peto OR 4.01, 95% CI 0.82 to 19.56; PR/ couple swim-up 15%, gradient 20%). There was no significant difference in the miscarriage rate (MR) per couple between either of the three treatment groups in the one trial reporting this outcome (MR/ couple swim-up 0%, gradient 10.3%, wash 0%. MR/ pregnancy gradient 30.3%). There was no statistically significant difference in the multiple pregnancy rate (MPR) per couple between either of the three treatment groups in Dodson 1998-I/ II (MPR/ couple swim -up 0%, gradient 0%, wash 6.3%). One triplet pregnancy was recorded. Fifteen studies comparing semen parameters after processing were included. Two studies were included in the meta-analysis, we were not able to pool results. REVIEWERS' CONCLUSIONS: There is insufficient evidence to recommend any specific preparation technique. Large high quality randomised controlled trials, comparing the effectiveness of a gradient and/ or a swim-up and/ or wash and centrifugation technique on clinical outcome are lacking. Further randomised trials are warranted. Results from studies comparing semen parameters may suggest a preference for gradient technique, but firm conclusions cannot be drawn and the limitations should be taken into consideration.

Centrifugation, Density Gradient↗

Insemination-induced histolysis of the flight musculature in fire ants (Solenopsis, spp.): an ultrastructural study (1).

The fine structure of the insemination-induced flight muscle degeneration (histolysis) in the queen fire ant (Solenopsis spp.) has been investigated. Within 2 hours post-insemination, degenerative changes are detectable in the most peripheral fasciculi of the fibrillar flight muscle. Histolysis proceeds internally with time. Myofibril (sarcomere) dissolution begins with myofilament breakdown and continues until only free Z-line material remains. The latter subsequently disappears leaving, at the terminal stages, only nuclei, lamellar bodies (myelin figures), and tracheoles as residual myoid elements. Lysosomes and/or phagocytes do not appear to play a primary role in the initiation and continuance of this process.

Animals↗

A simple technique to increase success rate of artificial insemination.

A simple technique to increase the success rate of artificial insemination is described: most of the cervical mucus is aspirated prior to the endocervical injection of the husband's or donor's semen. The bulk of seminal fluid is kept in place; its back flow into the vagina is prevented by gentle pressing of the syringe by the patient. Sixty-six women underwent treatment by this technique. The pregnancy rate was significantly higher, and fewer cycles of treatment per pregnancy were required, as compared to 65 women inseminated by simple endocervical injection. No side effects except mild transitory abdominal pain were encountered.

Cervix Mucus↗

Reproductive morphology of Brittanichthys axelrodi (Teleostei: Characidae), a miniature inseminating fish from South America.

Light and electron microscopy were used to investigate the morphology of reproductive characters in a characid fish, Brittanichthys axelrodi. Spermatozoa were found in ovaries of females, thereby confirming insemination in this species. Bony hooks can be found on the fourth unbranched ray and branched rays 1-4 of the anal fin and the unique sigmoidally-curved ray of the caudal fin in mature males. Testes have three distinct regions: an anterior spermatogenic region, an aspermatogenic middle region lined with a simple squamous epithelium and used for storage of mature spermatozoa, and a posterior region of coiled chambers lined with a high simple cuboidal epithelium. The most posterior region appears to be instrumental in the formation and storage of spermatozeugmata, unencapsulated sperm packets. Thus far, this tripartite testis morphology is unique among characids. The mature spermatozoon has an elongate nucleus ( approximately 5 microm in length). A striated rootlet originates at the anterior end of the distal centriole and continues to the anterior tip of the cell. The striated rootlet wraps around the entire ventral area of the anterior part of the nucleus and appears to continue around the anterior tip of the nucleus and down the dorsal side as electron-dense material. Several large, spherical mitochondria ( approximately 0.6 microm in diameter) with lamellar cristae overlap the posterior end of the nucleus and continue beyond together with the cytoplasmic collar that contains the flagellum which lacks axonemal fins. Each spermatozeugma is lanceolate in shape when sectioned mid-sagitally, with the core staining positively for mucopolysaccharides. In both sexes, the gonopore opens posterior to the anus, with the urinary pore having a separate opening posterior to the gonopore. Bands of skeletal muscle were found in the area of the male gonopore. These morphological features are likely linked to the reproductive mode of insemination, a trait that is so far as known, relatively rare among teleost fishes, but is proving increasingly frequent among certain groups of characid fishes.

Animals↗

Subzonal insemination with a single spermatozoon using manipulation assisted sperm adhesion onto the ooplasmic membrane in mouse ova.

A simple and successful method of microinjection of a single spermatozoon under the zona pellucida of a mouse oocyte has been developed. A characteristic of this method is that the tip of the sperm injection needle pierces the zona pellucida without touching the ooplasmic membrane. All the ova (277) used for this series of experiments had normal morphology after the injection procedure. Spermatozoa preincubated in culture medium for capacitation and those treated with ionophore A23187 for induction of acrosome reaction were used. In combination with some of these injections, a manipulation assisting the adhesion of the sperm head onto the ooplasmic membrane was employed. The fertilization rate (67.3%) of the ova injected with the ionophore-treated sperm using the sperm-adhesion treatment was significantly higher (P less than 0.005) than that obtained by the injection of the preincubated sperm without applying the adhesion treatment (23.6%). All three of the recipients that received the 24 fertilized ova became pregnant and gave birth to 11 offspring (45.8%). The inseminations performed with the sperm-adhesion treatment using the immotile sperm from the preincubated population and/or those from the ionophore-treated population did not result in fertilization in any case. These results suggest that the fertilization rate of subzonal insemination with motile ionophore-treated sperm can be improved by applying the sperm-adhesion treatment and that sperm motility might be involved in the establishment of fertilization, even after the adhesion of the sperm head with the mouse ovum membrane.

Animals↗

Incorporation and fate of specific sperm plasma membrane components following insemination as revealed by ultrastructural immunocytochemistry.

Studies have been carried out to 1) further characterize sperm specific plasma membrane polypeptides (33 and 35 kDa) that are recognized by a monoclonal antibody previously described (Longo, 1989) and 2) follow the incorporation and dispersal of these proteins within plasmalemmae of monospermic and polyspermic sea urchin (Arbacia punctulata) eggs and oocytes utilizing immunocytochemical methods at the ultrastructural level of observation. Only sperm labeled when incubated with monoclonal antibody to the 33 and 35 kDa proteins followed by colloidal gold-tagged second antibody. Colloidal gold label was observed on the egg plasma membrane immediately after gamete membrane fusion; the amount and extent of label, i.e., the distance from the site of sperm incorporation, increased with time postinsemination. By 20 min postinsemination approximately one hemisphere of the inseminated egg/oocyte was associated with label. The expanding distribution of colloidal gold label on inseminated eggs and oocytes vs. time reflects the free diffusion of 33 and 35 kDa sperm surface proteins among egg/oocyte plasma membrane components. Label was also found in forming endocytotic vesicles, suggesting that sperm plasma membrane proteins may be internalized.

Animals↗

Identification of male-factor semen samples prior to insemination and in vitro fertilization.

Semen analyses carried out as part of the clinical in vitro fertilization or intrauterine insemination protocols provide important information that determine the type of clinical treatment of the male partner and the sperm processing method. It is postulated that the sperm of male-factor patients cannot survive hypoosmotic stress conditions because of defective sperm membrane function. To test this, 0.1 ml of semen from each of 102 patients was placed in 1.0 ml of 150 mosmol/liter eosin citrate fructose solution and incubated for 30 min at 37 degrees C. The percentage viability of the sperm cells was then determined. The results indicated that patients with two or more abnormal semen parameters had a significantly lower percentage viability while in the hypoosmotic solution (40.6 +/- 4.7%), in contrast to non-male-factor patients (69.0 +/- 1.6%). Donor sperm (N = 32) serving as controls (73.3 +/- 2.1%) had a viability in hypoosmotic solution similar to that of non-male-factor patients. The data suggest that sperm of male-factor patients are less able to survive the hypoosmotic stress conditions as shown by the percentage viability in hypoosmotic solution and emphasize the importance of using less stressful sperm processing methods for in vitro fertilization or insemination in these patients.

Cell Membrane↗

Success of intrauterine insemination using cryopreserved donor sperm is related to the age of the woman and the number of preovulatory follicles.

OBJECTIVE: Our objective was to assess parameters associated with a successful outcome of intrauterine insemination (IUI) using cryopreserved donor sperm. DESIGN: We analyzed 750 consecutive donor IUI cycles undertaken by 363 women in an assisted conception clinic. The main outcome measure was clinical pregnancy. RESULTS: IUI was performed in 94.7% of the 750 IUI treatment cycles commenced and 180 clinical pregnancies occurred. The clinical pregnancy rate per cycle was 26.4%. The rate was significantly related to the patient's age (30.5% for age < or = 35 years and 18.1% for age > 35 years; P < 0.006) and whether there was one or more than one preovulatory follicles [20.9, 34.4, and 31.5% for one, two, and three or four follicles with a mean diameter of 14 or more mm at the time of human chorionic gonadotropin (hCG) administration; P = 0.006]. Two to four preovulatory follicles were present in 12.6% of the natural cycles, 43.6% of clomiphene citrate or tamoxifen, and 59.9% of gonadotropin stimulated cycles. The difference in the number of preovulatory follicles between stimulated and unstimulated cycles was highly significant (P < 0.0001). Pregnancy rates were 29.9% in gonadotropin-stimulated cycles, 23.6% in clomiphene citrate- or tamoxifen-stimulated cycles, 23.6% in clomiphene and 20.1% in unstimulated cycles. The difference in pregnancy rates between gonadotropin-stimulated and natural cycles was significant (P = 0.038). Cycle fecundity rates were not significantly affected by the number of previous treatment cycles, duration of infertility, gravidity and parity of the patient, presence of a spontaneous luteinizing hormone (LH) surge before the administration of hCG, or number of motile sperm in the insemination specimen. CONCLUSIONS: Success of IUI using cryopreserved donor sperm is related to the age of the women and whether there is one or more than one preovulatory follicles.

Cryopreservation↗

Accuracy of pregnancy/non pregnancy diagnosis in zebu and crossbred cattle and Murrah buffaloes by milk progesterone determination post insemination.

Milk samples collected on days 0, 6, 20, 22 and 24 post insemination from Sahiwal and Tharparkar (zebu) cows, Karan Swiss (Sahiwal x Brown Swiss) and Karan Fries (Tharparkar x Holstein Friesian) crossbred cows and Murrah buffaloes were analysed for progesterone by enzyme-linked immunosorbent assay to determine the accuracy of pregnancy and non pregnancy diagnosis in each of the 3 groups of animals. The accuracy of pregnancy diagnosis was markedly improved in cows when the milk progesterone values on days 20, 22 and 24 were considered together, and a further improvement when the levels on days 0, 6, 20, 22 and 24 were considered together particularly in crossbred cows. In contrast, buffaloes exhibited the highest degree of accuracy from samples collected on day 24, and improvement in accuracy was minimal when values were considered for all 5 days, viz. 0, 6, 20, 22 and 24 post insemination. Overall, the accuracy of positive pregnancy diagnosis was greatest in zebu cows (90.9%) followed by crossbred cows (86.6%) and Murrah buffaloes (76.5%) when milk progesterone values on all 5 days were considered. The accuracy for negative pregnancy diagnosis was 100% on all days. The results suggest that there is a greater variability in oestrous cycle length in cattle than in buffaloes, and embryonic mortality is greater in buffaloes than in cattle.

Animals↗

Subzonal insemination (SUZI) or in vitro fertilization (IVF) in microdroplets for the treatment of male-factor infertility.

PURPOSE: The results of subzonal insemination (SUZI) and in vitro fertilization with microdroplet insemination used in couples with male-factor infertility are presented. RESULTS: The total fertilization rate was 17.4% for SUZI (n = 89) and 49.3% for microdroplet IVF (n = 100). The fertilization rate for standard IVF (n = 510), not including any male-factor infertility and performed during the same period, was 73.2%. The "take-home baby rate" per started cycle and per embryo transfer (ET), respectively, was 10 and 17.6% for SUZI and 20 and 24.7% for microdrop IVF. For standard IVF these figures were 27 and 31.7%. CONCLUSION: It was concluded that microdroplet IVF can be used with good results in cases of moderate male-factor infertility. The normal (2PN) fertilization rate with the SUZI technique was only 15.1%. However, despite the low fertilization rate, SUZI should be considered when dealing with severe male-factor infertility.

Fertilization in Vitro↗

Controlled ovarian hyperstimulation and intrauterine insemination for infertility associated with endometriosis: a retrospective analysis.

OBJECTIVE: To evaluate the efficacy of controlled ovarian hyperstimulation and intrauterine insemination for infertility associated with endometriosis. MATERIAL AND METHODS: A retrospective analysis of 260 patients with the only diagnosis of endometriosis, or male factor, or tubal factor, or unexplained infertility were performed: a total of 56 patients with different stages of endometriosis, a control group consisting of 38 patients with male factor infertility, a group of 26 patients with tubal factor infertility and a group of 140 patients with others (unexplained infertility, ovulation disorders, cervical factor). Pregnancy rate, hormone levels, endometrial thickness and number of follicles were analyzed. RESULTS: Clinical pregnancy rates per patient were similar between endometriosis, male factor, tubal factor, and others including unexplained infertility, ovulation disorders and cervical factor groups (10.7%, 5.4%, 11.5%, 17.9%, respectively; p>0.05). Clinical pregnancy rates per patient were not effected between the 2 subgroups of endometriosis as minimal to mild and moderate to severe [5.1% (2/39) versus 23.5% (4/17), p=0.19]. CONCLUSION: Endometriosis did not affect the clinical pregnancy rate per patient compared to the other infertility factors. Endometriosis of various stages have no effect on the success of controlled ovarian hyperstimulation combined with intrauterine insemination.

Adult↗

Clomiphene citrate induced ovulation and intrauterine insemination: effect of timing of human chorionic gonadotropin injection in relation to the spontaneous LH surge on pregnancy rates.

PURPOSE: To determine the optimal time for administration of human chorionic gonadotropin in clomiphene citrate induced intrauterine insemination cycles. METHODS: A retrospective analysis of 171 consecutive cycles was performed. An increase in luteinizing hormone level >100% over the mean of the preceding two days was defined as luteinizing hormone surge. Human chorionic gonadotropin was given in preparation for intrauterine insemination based on the follicle size and estradiol level prior to surge in 85 cycles (Group A), with the spontaneous surge in 64 cycles (Group B) and not given in 22 cycles (Group C) due to high luteinizing hormone levels. RESULTS: The overall pregnancy rate per cycle was 18.1% (31/171), 15.2% (Group A), 20.3% (Group B) and 22.7% (Group C), (p > 0.50). CONCLUSION: Although there may be physiological reasons to propose that timing the human chorionic gonadotropin to the luteinizing hormone surge will improve the success rate, they were not demonstrated.

Adult↗

The significance of antral follicle count in controlled ovarian stimulation and intrauterine insemination.

PURPOSE: This retrospective study evaluated the role of antral follicle count (AFC) in predicting ovarian response and successful outcome of stimulated insemination cycles. METHODS: One-hundred and fifty infertile women with bilateral patent tubes receiving a standard regime of human menopausal gonadotrophin (HMG) in their first cycle were evaluated. Multiple regression analysis was used to evaluate the effects of different parameters on ovarian responses and multiple logistic regression analysis was applied to determine which parameters gave the maximum discrimination to predict clinical pregnancy. RESULTS: Body mass index was the only significant parameter to predict the number of follicles > or =14 mm whereas AFC was the only significant parameter to predict the HMG duration. Only the number of follicles > or =14 mm significantly improved the chance of clinical pregnancy with an odds ratio of 1.8. CONCLUSION: AFC was related to the HMG duration but was not predictive of number of follicles and clinical pregnancy of stimulated insemination cycles.

Adult↗

Comparing the pregnancy rates of one versus two intrauterine inseminations (IUIs) in male factor and idiopathic infertility.

PURPOSE: To compare the effectiveness of double IUI with single IUI in male factor and idiopathic infertility patients undergoing controlled ovarian hyperstimulation (COH). METHODS: A prospective randomized study of 1257 COH-IUI cycles was performed. Total 1270 patients with mild or moderate male factor infertility (n = 776) and idiopathic infertility (n = 494) were enrolled in this study, and 767 with male factor infertility and 490 with idiopathic infertility completed clomiphene citrate (CC)/human menopausal gonadotrophin (hMG) COH cycles and IUI. Categorized on the basis of the causes of infertility (male factor, M; idiopathic infertility, I), patients were randomized into one of the following groups: Single IUI group (M(1)/I(1)) received single insemination 34 h post hCG administration, and double IUI group (M(2)/I(2)) received two inseminations 18-24 and 36-48 h post hCG injection, respectively. RESULTS: The overall pregnancy rates were 15.43%. Pregnancy rate for single and double IUI group was 11.06 and 19.87% (p < 0.05), respectively. There was a significant statistical difference in cycle fecundity between M(1) and M(2) group (11.34% vs. 24.93%, p < 0.05), and between I(2) and M(2) group (11.93% vs. 24.93%, p < 0.05), but there was no significant difference between I(1) and I(2) (10.53% vs. 11.93%, p > 0.05). CONCLUSIONS: Double IUI increases the pregnancy rate significantly in patients with male factor infertility, and single IUI acts as efficient as double IUI in patients with idiopathic infertility.

Female↗

A randomized comparison of intrauterine and intraperitoneal insemination in the treatment of infertility.

OBJECTIVE: To compare the pregnancy rates (PRs) between intrauterine insemination (IUI) and intraperitoneal insemination (IPI) in women receiving comparable ovarian stimulation in couples who were infertile for various infertility conditions. METHOD: Fifty-four couples were treated. Of these, 25 were diagnosed as having unexplained infertility, 21 as having male infertility and 8 as having cervical mucus hostility. RESULT: Nine pregnancies occurred with 89 cycles of IUI and 6 pregnancies with 85 cycles of IPI, giving an overall PR of 28%. Twelve of the 15 pregnancies occurred during the first treatment cycle and three during the second treatment cycle. There was no significant difference in the (PRs) between the IUI and IPI (P > 0.25). CONCLUSION: Both procedures combined with superovulation proved to be simple, inexpensive safe and worthwhile for infertile couples with unexplained infertility, male infertility and cervical mucus hostility.

Adult↗

Postcoital-voiding insemination: technique for patients with retrograde ejaculation and infertility.

Three patients with infertility secondary to retrograde ejaculation demonstrated motile sperm in their urine after coitus. These patients were instructed to alkalinize their urine and void directly into the vagina after intercourse. The technique was called "postcoital-voiding insemination." Three pregnancies have resulted. The technique appears to be a good alternative to bladder catheterization and artificial insemination.

Adult↗

Insemination of HIV-negative women with processed semen of HIV-positive partners.

Many HIV-discordant couples want to have children so much that they are willing to abandon condom-protected sexual intercourse irrespective of the risks. Previous testing in our laboratory showed that gradient centrifugation followed by a swim-up procedure effectively removed HIV-1-infected cells from the semen of HIV-seropositive men. 85 HIV-discordant couples were screened for fertility; 29 women were found suitable for a timed insemination course with the processed semen of their HIV-seropositive partner. None of the inseminated women seroconverted, and 17 pregnancies were achieved in 15 women. All 10 babies born to these mothers remain HIV seronegative. The findings may help in the counselling of such couples and also give them hope of having healthy babies.

Centrifugation, Density Gradient↗