Caleb A. Awoniyi

Reproductive sequelae in female rats after in utero and neonatal exposure to the phytoestrogen genistein

OBJECTIVE To determine reproductive sequelae in female rats after in utero and lactational dietary exposure to genistein. DESIGN Experimental animal study. SETTING University laboratory. ANIMAL(S) Sprague Dawley rats. INTERVENTION(S) Pregnant rats were fed control rat chow or rat chow incorporated with genistein (approximately 50 microg/d) beginning on day 17 of gestation and continuing until the end of lactation (postpartum day 21). Genistein-exposed female pups were divided into two groups on day 21. One group continued to receive a genistein-added diet (G70); the other group was changed to a control diet (Ex-G). At necropsy (days 21 and 70), blood and reproductive tissues were collected. MAIN OUTCOME MEASURE(S) Serum levels of gonadotropins and gonadal steroids and histopathologic examination of the ovaries. RESULT(S) The weight of the ovaries and uterus and serum levels of E2 and progesterone in genistein-exposed rats on day 21 (G21) were significantly reduced compared with control rats. On day 70, serum levels of E2, progesterone, LH, and FSH were similar in all groups. Atretic follicles and secondary interstitial glands were more common in G70 and Ex-G rats compared with control rats. Cystic rete ovarii was observed in some G70 and Ex-G rats. CONCLUSION(S) Our data indicate that in utero and lactational exposure to dietary genistein adversely affects reproductive processes in the adult female rat.

Effects of chronic dietary exposure to genistein, a phytoestrogen, during various stages of development on reproductive hormones and spermatogenesis in rats

Development, hormonal, and gametogenic parameters were evaluated in male progeny following chronic dietary exposure to the phytoestrogen genistein. Twenty pregnant rats were fed a diet containing genistein (50 μg/d) from d 17 of gestation, and 12 were fed a control diet without genistein. Four litters each of control and genistein-fed rats were euthanized on d 21. The remaining pups were weaned on d 21 and only male rats were used in this study. On d 21, eight litters of genistein-fed rats were placed on control diet (gestational and lactational exposure alone [GL-G]), and the remaining eight continued on genistein diet (lifelong exposure group [LL-G]). These rats were euthanized (four litters/group) on d 70 or 130 of life. Serum testosterone, which was slightly reduced in genistein-exposed rats on d21, did not differ among treatment and control groups on d 70 and 130. Serum luteinizing hormone of genistein-exposed rats was reduced on d 21 and 130, but not on d 70. Serum follicle-stimulating hormone did not vary among groups at any age. Treatment-related effects of dietary genistein were not observed on the weights of the testes of 21-d-old rats. Except for a slight decrease in testis weight of GL-G rats at 130 d, no significant effect of dietary exposure was observed on the weight of the testes in any other group. However, epididymal weights were significantly reduced in both treated groups at d 130. Testicular sperm count (on d70 as well as 130) also was not affected in GL-G or LL-G rats. We conclude that gestational plus lactational exposure to genistein and subsequent dietary exposure to genistein have no adverse effects on gametogenic function in male rats.

Assisted hatching does not enhance IVF success in good-prognosis patients

AbstractPurpose: The role of assisted hatching in good-prognosis IVF patients was evaluated in a prospective, randomized, controlled pilot study, which was followed by a retrospective observational series. Methods: After assisted hatching was proved successful in a mouse embryo study, 20 good-prognosis IVF patients were randomly assigned to either assisted hatching (13) or no assisted hatching (7; the controls). Following this series, 27 good-prognosis IVF patients were retrospectively evaluated to determine the outcome with assisted hatching. Results: In the prospective study, clinical pregnancies resulted from 3 (23%) of 13 patients in the hatching group, compared to 3 (43%) of 7 in the control group. Implantation rates were similar: 9.6% in the hatching group and 10.7% in the controls. In the retrospective series, the 11.1% implantation rate with assisted hatching was significantly less than the 42.9% implantation rate seen with traditional IVF. Conclusions: Implantation and pregnancy rates are high in young women undergoing traditional IVF. Assisted hatching is not beneficial in these patients.

Hydrosalpinx treated with extended doxycycline does not compromise the success of in vitro fertilization

OBJECTIVE To determine if extended treatment with doxycycline before and after an in vitro fertilization (IVF) procedure can minimize the detrimental effect of a hydrosalpinx. DESIGN Retrospective analysis. SETTING University IVF program. PATIENT(S) Patients undergoing IVF, including 17 with a hydrosalpinx, 25 with adhesions or proximal tubal occlusion, and 22 with endometriosis or unexplained infertility. INTERVENTION(S) Women with a documented hydrosalpinx were prescribed doxycycline 100 mg twice daily starting 1 week before expected retrieval and continued until 6 days after retrieval. No antibiotics were prescribed in the other groups. MAIN OUTCOME MEASURE(S) Implantation rates and IVF outcomes. RESULT(S) Implantation rates were 30% for the doxycycline-treated group of patients with a hydrosalpinx, 27% for the group with tubal occlusion/adhesion, and 24% for the group with endometriosis or unexplained infertility. Eight (47%) of 17 patients with a hydrosalpinx had a live birth, compared with 11 (44%) of 25 for the group with tubal occlusion/adhesion and 12 (55%) of 22 for the group with endometriosis/unexplained infertility. There were no differences between the groups in patient age, number of oocytes retrieved, fertilization rate, or number of blastomeres of the transferred embryos. CONCLUSION(S) No detrimental effect of a hydrosalpinx was evident for patients treated with extended doxycycline. Tremendous cost savings can be realized if treatment with 2 weeks of an inexpensive antibiotic provides outcomes comparable to surgical correction of a hydrosalpinx before IVF.

Neonatal exposure to coumestrol, a phytoestrogen, does not alter spermatogenic potential in rats

The objective of this study was to determine the effects of neonatal exposure to phytoestrogens on male reproductive function as adults. Male rats were injected either with 100 μg coumestrol or DMSO (controls) daily during their first 5 d of life. Pituitary gland, testes, sex accessory organs, and blood were collected on d 60 of life. Serum testosterone, LH, and FSH levels were determined by RIA. Levels of steady-state mRNA for gonadotrophin subunits (LHβ and FSHβ were determined by Northern blot analysis and quantified by a scanning densitometer. Coumestrol had no effect on weights of testes and sex accessory organs, or sperm count. Similarly, there were no significant differences among serum concentrations of testosterone, LHβ and FSH of coumestrol-treated rats and those of controls. Whereas steady state levels of LHβ mRNA in coumestrol-treated rats did not differ from those of controls, steady state levels of FSHβ mRNA increased (37%) in treated animals. However, the augmented FSHβ mRNA expression in coumestrol-treated rats did not negatively affect reproductive potential in male rats. We conclude that neonatal exposure to coumestrol does not alter reproductive organ structure or spermatogenic potential in male rats.

Evaluation of synthetic serum substitute versus serum as protein supplementation for mouse and human embryo culture.

Purpose: Our purpose was to determine the effect of Synthetic Serum Substitute (SSS) versus serum supplementation on fertilization rates and subsequent development of embryos from patients undergoing IVF.Procedure: Experiment I compared the effects of SSS to human serum on mouse embryo development. Two hundred one-cell B6D2F1 mouse embryos were cultured in 100-µl droplets of human tubal fluid (HTF) containing either (1) no protein (control;n=37), (2) 15% serum from women with tubal infertility (n=44), (3) 15% serum from women with endometriosis (n=49), (4) 15% fertile donor serum (n=33), or (5) 15% SSS (n=37). Experiment II compared the effects of SSS to human serum on the development of embryos from patients undergoing IVF. Thirty-three women were included in this study. A total of 371 oocytes was cultured in HTF containing either (1) maternal or donor serum (n=140) or (2) 15% SSS (n=231). Embryo development was evaluated 48 hr after fertilization.Results: In Experiment I, the rate of blastocyst development was evaluated at 48, 72, and 96 hr of culture. Sixty-four and nine-tenths percent of embryos cultured in SSS were morulae at 48 hr of culture (versus 5.4, 0, 8.2, and 6.1 in Groups 1, 2, 3, and 4, respectively). By 72 hr, 29.7% of these embryos had developed into blastocysts (versus 0, 0, 8.2, and 3.0, for Groups 1, 2, 3, and 4, respectively). This percentage increased to a total of 83.7 after 96 hr (versus 27.0, 20.4, 38.8, and 39.4 for Groups 1, 2, 3, and 4, respectively). Forty-three and two-tenths percent of the blastocysts cultured in SSS had hatched from their zonae by 96 hr. With the exception of Group 5, which had a rate of 9.1%, embryo hatching was not observed in any of the groups at the termination of culture (96 hr). In Experiment II there were no differences in cell stage or quality of human embryos cultured in SSS or serum, but fertilization rates tended to be better (P=0.07) for oocytes inseminated in media containing SSS (70.0%, vs 55.0% for serum).Conclusions: SSS appears to be a superior protein source for mouse embryo growth and is as good as serum from fertile donors in promoting in vitro human embryo development.

Immunoneutralization of gonadotropin-releasing hormone and subsequent treatment with testosterone Silastic implants in rats: an approach toward developing a male contraceptive*

STUDY OBJECTIVE To determine the extent to which increasing doses of exogenous testosterone (T) administered via Silastic implants can restore spermatogenesis and fertility to rats made azoospermic by active immunization against gonadotropin-releasing hormone (GnRH). DESIGN Male rats were made azoospermic by active immunization against GnRH. Increasing doses of exogenously administered T (via Silastic implants) were administered for 8 weeks, and testicular sperm concentration and ability to impregnate female rats were evaluated. SETTING Reproductive Endocrinology Laboratory, Department of Obstetrics and Gynecology, University of Colorado Health Sciences Center, Denver, Colorado. ANIMALS Sexually mature male Sprague Dawley rats (SASCO, Omaha, NE). RESULTS Suppression of gonadotropins and azoospermia was achieved by actively immunizing rats against GnRH. Testosterone was capable of restoring quantitatively complete spermatogenesis and fertility in GnRH-immunized azoospermic rats. This relationship was dose-dependent, as evidenced by the partial restoration of spermatogenesis and fertility observed in animals replaced with smaller T Silastic implants. CONCLUSION Gonadotropin-releasing hormone immunization and T-filled Silastic implants may provide a model to study isolated gonadotropin deficiency and for the development of a reversible male contraceptive.

Preprogrammed, unmonitored ovarian stimulation reduces expense without compromising the outcome of assisted reproduction☆

OBJECTIVE To determine if a novel, preprogrammed, unmonitored stimulation protocol could reduce the cost of assisted reproductive technology (ART) without compromising outcome or safety. DESIGN Prospective, nonrandomized study of unmonitored ART versus traditional monitoring. SETTING University ART program. PATIENT(S) Infertile women aged < 39 years, with a basal FSH level < 15 mIU/mL (conversion factor to SI unit, 1.00) and regular menstrual cycles, undergoing ART. INTERVENTION(S) Oocyte retrieval was performed at a predetermined time in 72 unmonitored cycles based on age and basal FSH level. No monitoring of any type was performed before retrieval. There were 86 monitored control cycles. MAIN OUTCOME MEASURE(S) The number of oocytes, and embryos; complications including ovarian hyperstimulation. RESULT(S) The total cost for unmonitored ART was significantly less than for monitored cycles. There was no difference between groups for patient age, number of oocytes obtained, or number of metaphase II oocytes. For non-male-factor patients, the number of oocytes fertilized, number of embryos transferred, and the clinical pregnancy rates were comparable. There was one case of severe hyperstimulation requiring hospitalization in the unmonitored group. CONCLUSION(S) This novel, unmonitored ovarian stimulation protocol provides ART at a significantly lower cost than is incurred with traditional monitoring, with no apparent compromise in outcome.

Application of the cavitron ultrasonic surgical aspirator (CUSA) * for gynecological laparoscopic surgery using the rabbit as an animal model † ‡

OBJECTIVE To study the potential application of the cavitron ultrasonic surgical aspirator (CUSA) in gynecological laparoscopic surgery using a rabbit animal model. DESIGN Twenty-six rabbits were prospectively randomized into two groups. Laparoscopically directed standard injuries were made on the randomly assigned horn and sidewall in all animals with the CUSA. Contralateral injuries were made with a contact neodymium-yttrium aluminum garnet (Nd:YAG) laser in group 1 and with bipolar cautery in group 2. Adhesion and inflammation scores were assessed for two animals in each group at 24, 48, and 72 hours, and seven animals in each group at 14 days. SETTING University animal research facility. MAIN OUTCOME MEASURES Adhesion and inflammation scores were compared between animals in the CUSA versus Nd:YAG study and the CUSA versus bipolar cautery at 14 days. RESULTS No significant difference in uterine or sidewall adhesion scores was noted between the CUSA versus Nd:YAG or the CUSA versus bipolar cautery. Bipolar cautery produced significantly less inflammation on the uterine horn compared with the CUSA (3.0 +/- 0.2 versus 5.3 +/- 0.7, P = 0.0001), but no difference in sidewall inflammation was noted between the CUSA compared with bipolar cautery. No difference in inflammation was observed between the CUSA and the Nd:YAG laser. CONCLUSIONS The bipolar cautery appears to be preferable to the CUSA for coagulation of uterine lesions, although dissection of the uterus is not possible with bipolar cautery. The CUSA and the Nd:YAG appear to be comparable for uterine horn dissection. Because the CUSA causes similar adhesion formation and tissue inflammation at the sidewall when compared with the Nd:YAG laser and bipolar cautery and may be less likely to damage blood vessels, ureters, or other collagen-rich tissues, the CUSA may represent a promising new surgical tool for laparoscopically directed peritoneal dissection.

Pronuclear Uterine Transfer Lowers In Vitro Fertilization Success

The ideal time to perform embryo transfer (ET) has not been established. A prospective, randomized study showed no difference in pregnancy outcomes when 2-, 3-, and 4-day transfers were compared (1). Furthermore, no difference in success rates were seen between transfer on days 3 or 5 in another study (2). Currently, most in vitro fertilization (IVF) programs transfer embryos on day 2 or 3 and perform assisted hatching when this approach is believed to be beneficial. With extended culture times, there is a risk of developmental delay of healthy embryos. Assisted hatching may help overcome artificial zona hardening in culture, but this procedure adds to the complexity and expense of IVF. Embryo development is almost always enhanced in the presence of coculture, and endometrial cells appear to provide a beneficial effect (3). Unfortunately, coculture also adds to the complexity and expense of IVF. Since embryo development is enhanced with endometrial coculture, we hypothesized that early embryo uterine transfer may enhance implantation and pregnancy rates with IVF. To test the benefit of early ET, we initiated a study to determine whether pronuclear uterine transfer 1 day after oocyte retrieval was more effective than transferring cleaving embryos on day 2.