Performing ICSI using an injection pipette with the smallest possible inner diameter and a long taper increases normal fertilization rate, decreases incidence of degeneration and tripronuclear zygotes, and enhances embryo development.

PURPOSE: To compare the efficacy of two types of injection pipette used for ICSI, one with a larger (5-7 microns) inner diameter and a shorter taper with that inner diameter, and another with the smallest (3-5 microns) possible inner diameter and a longer taper with that inner diameter. METHODS: Retrieved oocytes at metaphase II stage were injected using one of two types of injection pipette, in 33 and 94 cycles, respectively, in a total of 127 cycles in 108 patients. RESULTS: In comparison to the injection pipette with a larger (5-7 microns) inner diameter and a shorter taper with that inner diameter, the injection pipette with the smallest (3-5 microns) possible inner diameter and a longer taper with that inner diameter increased normal fertilization rate ((70 +/- 3.6)% vs. (86 +/- 2.2)%; P = .001; mean +/- SEM); decreased the incidence of degeneration ((14 +/- 2.4)% vs. (5 +/- 1.4)%; P = .001) and tripronuclear zygotes ((1.0 +/- 0.35)% vs. (0.1 +/- 0.21)%; P = .03); increased Day-2 diploid embryos ((69 +/- 3.7)% vs. (85 +/- 2.2)%; P = .001) and good-quality Day-2 diploid embryos ((67 +/- 4.0)% vs. (79 +/- 2.4)%; P = .03), all per injected oocyte; and increased the number of blastomeres per good-quality Day-2 diploid embryo ((3.0 +/- 0.21 vs. 3.8 +/- 0.12; P = .0003). CONCLUSIONS: Performing ICSI using an injection pipette with the smallest (3-5 microns) possible inner diameter and a longer taper with that inner diameter maximizes normal fertilization rate, minimizes the incidence of postinjection degeneration and tripronuclear zygotes, and enhances embryo development.

Exposure of human oocytes to endometrioma fluid does not alter fertilization or early embryo development.

BACKGROUND: Extensive endometriosis causes a mechanical disturbance in the pelvis leading to obstructive-type infertility. However, minimal or mild endometriosis is suspected to cause infertility, possibly through a humoral agent. Previous studies reported the presence of a factor in the serum of patients with endometriosis which reduced fertilization and early embryo formation in a rat IVF model. METHODS: In the present article, we report a comparison of oocytes exposed to endometrioma fluid and oocytes not exposed (controls) in the context of a human IVF setting. We have been in the practice of aspirating oocytes into prewarmed 60-ml syringes containing culture medium. We have shown previously that this technique reduces the length of oocyte retrieval without compromising success. In 14 women undergoing oocyte retrieval, we inadvertently entered an endometrioma. This resulted in retrieved oocytes that were either exposed or not exposed to endometrioma fluid. RESULTS: In contrast to previous reports, we found no difference in fertilization or early embryo development between the two groups. The fertilization rate for oocytes exposed to an endometrioma was 60%, versus 56% for controls. The good-quality embryo formation rate for oocytes exposed to an endometrioma was 45%, versus 46% for controls.

Intracytoplasmic sperm injection increased fertilization and good-quality embryo formation in patients with non-male factor indications for in vitro fertilization: a prospective randomized study.

OBJECTIVE: To compare the fertilization rate and formation of good-quality embryos with conventional IVF and ICSI in patients with non-male factor infertility. DESIGN: Prospective controlled study. SETTING: Infertility clinic. PATIENT(S): Thirty-five patients with non-male factor infertility. INTERVENTION(S): Retrieved sibling oocytes were randomly assigned to conventional IVF or ICSI. Of sibling oocytes assigned to ICSI, only metaphase II oocytes were injected with sperm. MAIN OUTCOME MEASURE(S): Fertilization rate and formation of good-quality embryos per retrieved oocyte. RESULT(S): Per retrieved oocyte, ICSI resulted in better fertilization rate compared with conventional IVF (71.3% [134 of 188] vs. 57.2% [107 of 187]). Per retrieved oocyte, ICSI also resulted in better formation of good-quality embryos at 48 hours after retrieval compared with conventional IVF (64.4% [121 of 188] vs. 47.1% [88 of 187]). CONCLUSION(S): In IVF patients with non-male factor infertility, subjecting some sibling oocytes to ICSI increased the fertilization rate and formation of good-quality embryos per retrieved oocyte. It also avoided the problem of total fertilization failure in almost all cases.

Recent discoveries in physiology of insulin-like growth factor-1 and its interaction with gonadotropins in folliculogenesis.

Ovarian follicular development is under the influence of gonadotrophins. The manner by which gonadotrophins achieve their role in cell replication and hormone production is through other specialized molecules. Insulin-like growth factor-1 (IGF-1) is considered to be one of the most important of these molecules. IGF-1 is present in relatively large amounts in the ovary and the IGF-1 gene is expressed in the graafian follicle. IGF-1 binding proteins modulate the action of IGF-1 and are influenced by gonadotrophins. Null mutations of genes encoding IGF-1 and type-1 IGF receptor have demonstrated the obligatory role of IGF-1 in folliculogenesis. It was proposed that IGF-1 may be an obligatory mediator of gonadotrophin-induced folliculogenesis and that the interaction is synergistic. From this point, one group embarked on a series of physiological studies to further elucidate the interaction of these molecules. We discovered that, according to circumstances, follicle stimulating hormone (FSH) may act with IGF-1 synergistically (as was postulated before), additively, independently, or even antagonistically. We discovered that granulosa cells must first be exposed to FSH and then they respond well to IGF-1. All previous studies of granulosa cells were done on mural granulosa cells. We subsequently showed that cumulus type of granulosa cells (adjacent to the oocyte) behave differently in their responses to gonadotrophins and IGF-1.

Induction of ovulation in postpartum suckled beef cows: a review.

Prolonged postpartum acyclicity in suckled beef cows reduces the calf crop, and causes economic loss to beef cattle producers. Once anterior pituitary LH stores have been replenished between Days 15 and 30 post partum in suckled beef cows, methods to initiate cyclicity include non-hormonal methods such as weaning of calves (either complete, temporary or partial), or exposure to bulls, and hormonal methods such as administration of GnRH (either single injection, intermittent injections, or continuous infusion), gonadotropins (eCG, FSH, hCG), and steroids (estrogens, anti-estrogens, and progestogens). Weaning is costly, reduces growth rate of weaned calves, and short cycles are common after weaning-induced ovulation. Exposure of cows to bulls is not practical and its effect is not predictable. Repeated injections of GnRH, or a single injection of hCG are not always effective; ovulation is always followed by a short cycle, and usually a return to acyclicity. Estrogens and anti-estrogens do not consistently shorten postpartum anestrus. Exogenous progestogens include intravaginal devices, such as controlled-internal drug release (CIDR) or progesterone-releasing intravaginal device (PRID), norgestomet implants, and the feed-additive melengestrol acetate (MGA). Administration of exogenous progestogens is more practical than, and offers more advantages over, other treatments to shorten postpartum acyclicity in suckled beef cows. Mimicking the short cycle after Week 3 post partum, by maintaining circulating progesterone at subluteal concentrations or circulating progestin at intermediate concentrations, extends the life-span and allows terminal maturation of the postpartum dominant follicle as in cyclic cows, by initiating endogenous GnRH and LH pulses. This is followed by an LH surge, ovulation and normal cycles. The benefit from using exogenous progestogens after Week 3 post partum in suckled beef cows is that ovulation is induced, cyclicity is initiated, the resulting CL has a normal life-span and function, and there is no need to change management, such as weaning of calves. We present a model for the induction of ovulation and initiation of cyclicity using exogenous progestogens after Week 3 post partum in suckled beef cows.

Postpartum acyclicity in suckled beef cows: a review.

Prolonged postpartum acyclicity in suckled beef cows is a source of economic loss to beef cattle producers. Duration of postpartum acyclicity is influenced by suckling status, nutritional status, calving season, age, and several other factors. Although uterine involution begins and ovarian follicular waves resume soon after parturition, dominant follicles of these waves fail to ovulate, due to a failure to undergo terminal maturation. As a result, postpartum anovulatory dominant follicles are smaller than the ovulatory follicles in cyclic cows. Failure of postpartum dominant follicles to undergo terminal maturation is due to absence of appropriate LH pulses, a prerequisite for follicular terminal maturation prior to ovulation. Absence of LH pulses early post partum is primarily due to depletion of anterior pituitary LH stores, although GnRH pulses are also absent during this period due to suckling. Following replenishment of LH stores between Days 15 and 30 post partum, absence of LH pulses is due to continued sensitivity of the hypothalamic GnRH pulse-generator to the negative feedback effect of ovarian estradiol-17beta, which results in absence of GnRH pulses. This negative feedback effect of estradiol-17beta is modulated by suckling which stimulates release of endogenous opioid peptides from the hypothalamus. As the postpartum interval increases, sensitivity of the GnRH pulse-generator to the negative feedback effect of ovarian estradiol-17beta decreases. This is followed by an increasing frequency of GnRH discharges and LH pulses, terminal follicular maturation, ovulation, and continued cyclicity. The first ovulation post partum is usually followed by a short cycle due to premature luteolysis because of premature release of PGF2alpha from the uterine endometrium, which is possibly intensified by the suckling-induced oxytocin release from the posterior pituitary. A model for the postpartum ovulatory acyclicity and for the resumption of cyclicity is presented.

The status of controlled prospective clinical trials for efficacy of intracytoplasmic sperm injection in in vitro fertilization for non-male factor infertility.

PURPOSE: Intracytoplasmic sperm injection (ICSI) of some sibling oocytes may have a beneficial effect in couples going through in vitro fertilization for causes of infertility not related to the male factor. Our purpose was to critically appraise the randomized controlled studies done in this area and arrive at some recommendations. METHODS: The four controlled trials done so far have utilized similar methodology, i.e., they randomly allotted sibling oocytes to ICSI versus standard insemination in patients going through in vitro fertilization and embryo transfer. RESULTS: In the first trial reported in 1995 there was no difference in fertilization rate, whereas the later trials reported in 1997, 1999, and 2000 showed improvement with ICSI that reached statistically significant level in the last two studies. CONCLUSIONS: Total fertilization failure of an in vitro fertilization cycle can be prevented and fertilization can be improved if half of sibling oocytes are subjected to ICSI.

Effects of granulosa cells, cumulus cells, and oocyte density on in vitro fertilization in women.

The objective of this study was to determine if in vitro fertilization (IVF) rate is affected by the diameter of oocyte-cumulus complex (OCC), by coculturing oocytes with autologous granulosa cells, or by increasing the oocyte density in culture medium. Women with previous fertility problems underwent the IVF program. In study 1, the diameter of OCC was graded on retrieval on a scale of 0-3: grade 0 = no cumulus at all; grade 1 = diameter of 75-100 microm; grade 2 = diameter of 125-150 microm; and grade 3 = diameter of 200-225 microm. In study 2, oocytes were cocultured with autologous granulosa cells. In study 3, oocytes were cocultured in groups of one, two, or four. OCCs with a grade > 1.5/3 resulted in a greater (P = 0.04) proportion of embryo/oocyte than did OCCs with a grade < 1.5/3 (0.91 +/- 0.05 vs 0.68 +/- 0.10; mean +/- SEM). Coculturing oocytes with autologous granulosa cells did not affect (P = 0.42) the proportion of embryo/oocyte (0.63 +/- 0.11 vs 0.74 +/- 0.07 in controls). Coculturing oocytes in groups of two or four in culture drop did not affect (P = 0.37 and P = 0.38, respectively) the proportion of embryo/oocyte (0.63 +/- 0.07 vs 0.73 +/- 0.08 in controls, and 0.73 +/- 0.08 vs 0.63 +/- 0.08 in controls, respectively). In conclusion, coculturing oocytes with autologous granulosa cells or increasing the oocyte density from 1 to 2 or 4 oocytes/culture drop, in the context of our study, did not affect rate of IVF and embryo formation. The diameter of OCC at retrieval may give some indication regarding its future fertilization and development. This diameter varies with the type of ovarian stimulation and the patient's age. This variation in diameter does not correlate with oocyte maturity.

Modification of follicular dynamics by exogenous FSH and progesterone, and the induction of ovulation using hCG in postpartum beef cows.

Follicular growth and ovulation in response to FSH, progesterone and hCG were evaluated in postpartum beef cows. In Experiment 1, on Day 21 post partum, cows received an injection of either saline (control; n = 6), FSH (200 mg; n = 6), or a PRID (n = 5) for 10 d. Both FSH and PRID prolonged maintenance of a dominant follicle (15.5 +/- 1.16 and 14.4 +/- 1.29 d, respectively, vs 8.4 +/- 1.22 d in control; P < 0.01), and increased the maximum diameter of the dominant follicle (14.0 +/- 0.91 and 16.4 +/- 1.01 mm, respectively, vs 10.9 +/- 0.95 mm in control; P < 0.05). The PRID-maintained dominant follicle ovulated in 60% of cows, followed by normal estrous cycles (vs 0% in control; P = 0.01), whereas the dominant follicle ovulated in 33% of FSH-treated cows (P = 0.08). The PRID regimen shortened the interval to first ovulation preceding a normal cycle and continued cyclicity (44 +/- 4.1 vs 60 +/- 4.4 d in control; P = 0.02). In Experiment 2, on Day 21 post partum, cows received either saline (control), saline + PRID, or FSH + PRID (n = 16/group). Sixty hours after PRID withdrawal, cows received either saline or hCG (1,500 IU, n = 8/treatment). The FSH + PRID regimen increased the number of large (> 10 mm in diameter) follicles (3.6 +/- 0.43 vs 1.9 +/- 0.39 in control; P = 0.005). Both PRID and FSH + PRID prolonged maintenance of the largest follicle (11.0 +/- 0.82 and 11.2 +/- 0.91 d, respectively, vs 8.7 +/- 0.81 d in control; P < 0.05). The PRID-maintained dominant follicle ovulated in 50% of cows, followed by normal estrous cycles. The FSH + PRID-maintained largest follicle had become atretic at PRID withdrawal and was anovulatory. The FSH + PRID + hCG regimen increased the incidence of ovulation preceding a cycle of normal duration and continued cyclicity (100 vs 50% in PRID; P = 0.03), and reduced the interval to first ovulation preceding a cycle of normal duration and continued cyclicity (38 +/- 6.5 vs 58 +/- 6.3 d in control; P = 0.04). The area under the progesterone curve during the induced cycle was reduced after (PRID +/- FSH) + hCG than after PRID +/- FSH (P = 0.002). These results indicate that PRID alone or with FSH/hCG has the potential to modify the dominant follicle and initiate cyclicity in postpartum beef cows.

Trucking stress at breeding does not lower conception rate of beef heifers.

The goal of this study was to determine whether 1 h of trucking stress before or after artificial insemination (AI) altered the conception rate of beef heifers. Estrus was synchronized in heifers with prostaglandin F(2alpha). The 3 treatment groups consisted of 1) AI (control heifers, n = 93); 2) Truck + AI (trucked for 1 h immediately before AI, n = 81); and 3) AI + Truck (trucked for 1 h immediately after AI, n = 82). All heifers were artificially inseminated by a single technician with semen from a single ejaculate. Blood samples were collected for cortisol measurement 1 h before AI, immediately before and after AI, and 1 h after AI in the AI (n = 6), Truck + AI (n = 9), and AI + Truck (n = 8) groups Pregnancy in heifers was confirmed either at slaughter or by palpation per rectum. Trucking before AI elevated (P < 0.01) serum cortisol concentrations. Artificial insemination alone increased (P < 0.01) serum cortisol concentrations in AI heifers. The increase in serum cortisol concentrations caused by trucking after AI was not significant (P > 0.05). Areas under the cortisol curves in Truck + AI heifers are greater (P < 0.05) than in AI heifers. The conception rates of AI heifers (50.5%), Truck + AI heifers (51.9%) and AI + Truck heifers (58.5%) are not different (P > 0.05). This study demonstrates that 1 h of trucking stress either before or after AI did not lower the conception rate of heifers.

[Survey of the reproductive features and insemination/mating results in full blood Arabian and Haflinger mare herds on stud farms in west and middle Anatolia]. / Erhebung von Fortpflanzungsmerkmalen sowie Besamungs/Bedeckungsergebnissen in Vollblutaraber- und Haflingerstutenherden in Gestüten in West- und Mittelanatolien.

Within a German-Turkish university partnership documentation of reproductive data of brood-mares was performed as a part project of the cooperation contract. In the study Arab, Haflinger and cross-breed mares were included. The mares mainly were housed in big studfarms and a smaller part was kept under small private farms. Almost three quarters of both the Arab and Haflinger mares exhibited an estrous length of 1-4 days, whereas the others showed a heat duration of a period of 5-10 days. In the same group of probands a mean length of sexual cycle of 18-24 days could be observed in 38.2% of the Haflinger. The majority of the other mares in both races documented a cycle duration of more than 25 days. The remarkable short duration of heat is discussed as caused by the sharp change from winter to spring environment conditions in that area. An explanation of the marked long mean cycle interval is difficult and might be caused by inadequate documentation of the data. Conception results and foaling data as 83.4% resp. 78.8% in the Arabs and 76.6% resp. 74.8% in the Haflinger mares are within the international standard. Conception rate and foaling data in the cross-breed (72.3% resp. 55.3%) are low compared with data (96.9% 70.8%) of Arab brood-mares kept under stud condition in that area. These data point out the necessity of improving pre- and postconceptional care of the mares under stud conditions as well as in private keeping.