Optimal pregnancy outcome in a minimal-stimulation in vitro fertilization program.

OBJECTIVE: We sought to provide a cost-beneficial approach to in vitro fertilization for infertile patients who could not afford the standard treatment with in vitro fertilization and to determine the optimal level of minimal ovarian stimulation to achieve acceptable pregnancy rates. STUDY DESIGN: We performed a retrospective cohort study of 216 patients who underwent "minimal stimulation" in vitro fertilization between January 1994 and December 1998. During the first half of this study, various minimal ovarian stimulation protocols were performed in our private, free-standing center for in vitro fertilization. More recently, more ovarian stimulation, including a 4-day protocol featuring gonadotropin-releasing hormone agonist flare (ultrashort flare), was used. Clinical pregnancy outcome, multiple gestation, complications, and maternal age were compared between the first and second halves of this study. RESULTS: The average ages of patients in the first half (phase 1) and the second half (phase 2) were similar, 32.4 +/- 0.3 versus 32.6 +/- 0.3 years, respectively. An average of 3.5 oocytes per retrieval was obtained in phase 1 versus 5.9 oocytes in phase 2. Failure to retrieve oocytes occurred in 3% of all cases. The mean number of embryos transferred per patient was 2.0 in phase 1 versus 2.4 in phase 2. In phase 1, 16.1% of patients failed to have viable embryos for transfer, in comparison with 9.7% in phase 2. The overall clinical pregnancy rate per retrieval was 16.9% in phase 1 versus 36. 6% in phase 2. Multiple gestation occurred in 5.0% of clinical pregnancies in phase 1 but increased to 33% in phase 2, with 9 sets of twins and 6 sets of triplets. The implantation rate was 9.3% for phase 1 versus 23.3% for phase 2. The clinical pregnancy rates per retrieval for phase 2 patients were 41.6% in women < or =34 years old and 25.6% for those > or =35 years old. No case of ovarian hyperstimulation syndrome was noted. CONCLUSIONS: Minimal ovarian stimulation in the setting of in vitro fertilization offers a cost-beneficial alternative to standard treatment with in vitro fertilization in infertile patients who are <35 years old and in women <40 years old who have adequate oocyte reserve. More stimulation improves outcome. Minimalstimulation in vitro fertilization provides an alternative for those patients who cannot afford standard in vitro fertilization or who are concerned with exposure to high dosages of fertility medications.

Purified human alpha-fetoprotein inhibits follicle-stimulating hormone-stimulated estradiol production by porcine granulosa cells in culture.

We have investigated the effects of purified alpha-fetoprotein (AFP) on follicle-stimulating hormone (FSH)-stimulated estradiol production by porcine granulosa cells in monolayer culture. Granulosa cells isolated from small follicles of prepubertal pigs were cultured for 2 days in 5% fetal bovine serum for attachment and incubated for 3 days in medium containing androstenedione and various treatments. The media were then collected and assayed for estradiol by radioimmunoassay. Human AFP significantly (P < 0.05) and dose-dependently inhibited FSH-stimulated estradiol production with 313 ng/ml AFP returning FSH-stimulated estradiol to basal levels; human serum albumin was without effect. AFP purified from either term cord blood or midtrimester amniotic fluid dose-dependently inhibited estradiol production stimulated by the combination of FSH and insulin-like growth factor-I. Furthermore, 125 ng/ml AFP inhibited estradiol production stimulated by cholera toxin, forskolin and cAMP. In contrast, extracellular accumulation of cAMP and progesterone production was not inhibited by AFP. These data indicate that physiological concentrations of purified AFP significantly and dose-dependently inhibit FSH-stimulated estradiol production by porcine granulosa cells in culture. Since AFP is known to augment growth factor-mediated cell growth, these data suggest that AFP inhibits differentiated functions (such as aromatase) while enhancing the proliferation of porcine granulosa cells.

The secretion of transforming growth factor-beta by bovine luteal cells in vitro.

Transforming growth factor-beta (TGF-beta), a multifunctional polypeptide growth factor, is produced by follicular cells in the ovary. However, there is little information indicating that TGF-beta is produced in the post-ovulatory follicle, i.e. the corpus luteum. In the present communication we present evidence that bovine luteal cells secrete large amounts of TGF-beta when maintained in moderate-term monolayer culture. Using TGF-beta 1 and TGF-beta 2 subtype-specific antibodies to neutralize the bioactivity it was found that 80-90% TGF-beta activity in luteal cell conditioned medium (LCCM) is due to TGF-beta 1, whereas < or = 10% TGF-beta activity in LCCM is due to TGF-beta 2. TGF-beta subtype nonspecific antibodies effectively and completely neutralized all TGF-beta activity in LCCM. The ratio of TGF-beta 1:TGF-beta 2 as estimated on the basis of neutralization studies was supported by visual observation of TGF-beta 1 and TGF-beta 2 protein bands on Western blotting. Using a modified and rapid mink lung epithelial cell bioassay and authentic TGF-beta to generate standard curves, the amount of TGF-beta secreted by luteal cells in vitro was quantitated. The concentration of luteal cell secreted proteins in the medium increased with time during 7 days of culture. Likewise, the TGF-beta concentration in LCCM increased during 7 days. To study the effect of duration of culture on the rate of TGF-beta secretion by luteal cells, conditioned medium was collected at 24 h intervals and replaced with fresh nutrient medium.(ABSTRACT TRUNCATED AT 250 WORDS)

The regulation of porcine theca cell proliferation in vitro: synergistic actions of epidermal growth factor and platelet-derived growth factor.

An important but poorly understood aspect of mammalian follicle development involves the regulation of theca cell proliferation. To investigate the premise that growth factors regulate theca cell proliferation, porcine theca cells were prepared by collagenase/DN'ase digestion of follicle linings after the removal of the granulosa cells and allowed to attach for 24 h. This method provided a monolayer of theca cells that had little if any granulosa cell contamination and which secreted high levels of androstenedione relative to granulosa cells during moderate-term culture (33-fold difference, P less than 0.01). In medium containing fetal calf serum (10%), theca cells were significantly more responsive to platelet-derived growth factor (PDGF) than epidermal growth factor (EGF) in terms of proliferation (13.4 +/- 0.2-vs. 7.0 +/- 0.1-fold increases relative to the initial cell count, P less than 0.05). This is in contrast to granulosa cells which were significantly more responsive to EGF than PDGF (7.1 +/- 0.1 vs. 4.0 +/- 0.2 fold-increases, P less than 0.05). Since serum has been shown to contain both EGF and PDGF, proliferation studies were performed using plasma-derived serum (PDS) which is growth factor restricted to examine more closely the direct effects of growth factors. In medium containing 0.25% PDS and within experiments, PDGF (1-25 ng/ml) stimulated theca cell proliferation in a dose-dependent manner (2.3-fold increase relative to controls, P less than 0.05) whereas EGF did not. EGF, however, markedly enhanced the proliferative action of PDGF (6.4-fold increase relative to controls, P less than 0.05). Insulin-like growth factor I and low density lipoprotein, factors which enhance markedly the proliferative effects of EGF and PDGF in terms of granulosa cell proliferation, exhibited only a modest synergistic effect with respect to EGF and PDGF upon theca cells (9.5-fold increase vs. a 6.4-fold increase above controls, P less than 0.05). Temporal studies in vitro indicate that theca cell proliferation is low during the first 3-day exposure to growth factors irrespective of treatment (a 2-fold increase over the seeding density). During the second 3-day exposure, however, theca cell proliferation increases 4- to 5-fold. The temporal pattern of theca cell proliferation stimulated by fetal calf serum supplemented with EGF or PDGF and PDS-containing medium supplemented with PDGF, EGF, insulin-like growth factor I, and low density lipoprotein is similar. These results suggest that PDGF is a major mitogen toward porcine theca cells and that EGF greatly enhances its activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Purified human alpha fetoprotein inhibits growth factor-stimulated estradiol production by porcine granulosa cells in monolayer culture.

Purified alpha fetoprotein (AFP) synergizes with transforming growth factor alpha (TGF alpha) and insulin-like growth factor I (IGF-I) to enhance proliferation of porcine granulosa cells (pGC) in primary culture, suggesting a role for AFP in the modulation of growth factor-mediated cell growth. TGF alpha stimulates basal estrogen production by pGC and is in fact more potent than FSH in these cells. In this study, we investigated the effects of AFP on growth factor-stimulated estradiol (E2) production by pGC. Basal production of E2 was not altered by the addition of AFP. AFP dose-dependently inhibited TGF alpha-stimulated E2 production with statistically significant inhibition observed with 2.5 micrograms/ml. We have previously shown that the mitogenic effects of AFP are maximized with TGF alpha+IGF-I. E2 production was even more sensitive to AFP inhibition when the two growth factors were combined. Human serum albumin (HSA; 10 micrograms/ml) was without effect. AFP did not interfere with the E2 RIA, affect the uptake of or display specific in vitro binding of the androgen substrate. Furthermore, human AFP and HSA did not exhibit specific in vitro binding of E2, in contrast to purified rat AFP (positive control). These data indicate that physiological concentrations of purified AFP significantly and dose-dependently inhibit growth factor-stimulated E2 production by pGC in culture. Since AFP is known to increase TGF alpha+IGF-I mediated cell growth, these data suggest that AFP may be inhibiting the differentiated function (steroidogenesis) of pGC while enhancing the proliferation of these cells.

A novel mechanism for the induction of aromatase in ovarian cells in vitro: role of transforming growth factor alpha-induced protein tyrosine kinase.

In the developing follicle, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are the primary stimulators of steroidogenesis in granulosa and theca cells. The steroidogenic actions of both these gonadotropins are largely if not exclusively mediated through cAMP. Previous studies have shown that EGF and TGF alpha do not affect basal estrogen production but attenuate FSH-stimulated estrogen production in vitro in rat granulosa cells. Here we present evidence that TGF alpha stimulates basal estrogen production in prepubertal porcine ovarian granulosa and theca cells in culture under defined conditions. In granulosa cells, TGF alpha is more potent than FSH in stimulating estrogen production. LH does not stimulate estrogen production in prepubertal porcine theca cells but rather attenuates that stimulated by TGF alpha. Treatment of follicular cells with genistein (inhibitor of protein tyrosine kinase) attenuates TGF alpha-induced estrogen biosynthesis suggesting that the action of TGF alpha is mediated through protein tyrosine kinase. These studies provide evidence for an alternative cAMP-independent and TGF alpha-induced tyrosine kinase-dependent mechanism for the induction of ovarian aromatase.

Human mammary tumor cell proliferation: primary role of platelet-derived growth factor and possible synergism with human alpha-fetoprotein.

Human mammary medullary carcinoma cells (passages 16 to 21) were cultured for 2 days to allow for attachment, followed by 6 days of culture in either fetal calf serum, human cord blood, human amniotic fluid, or growth factors in the presence or absence of purified human alpha-fetoprotein (AFP). When growth factors were tested alone, only platelet-derived growth factor produced a significant increase in cell proliferation. Although up to 40% amniotic fluid had no effect on cell proliferation, human cord blood was two-fold more potent than fetal calf serum at similar concentrations. The addition of 10 ng/ml of platelet-derived growth factor increased the proliferative activity of human cord blood 1.5- to 2.5-fold. Ablation of endogenous AFP by affinity chromatography reduced the proliferative activity of cord blood by 75%. Similarly, the mitogenic activity of cord blood plus platelet-derived growth factor was reduced by 56% when AFP was removed. Purified AFP dose-dependently enhanced the proliferative activity of platelet-derived growth factor. This synergistic effect was specific for platelet-derived growth factor. We conclude that platelet-derived growth factor is a major growth factor controlling the proliferation of these tumor cells and that AFP may enhance growth factor proliferative activity and human mammary tumor growth.

The production of transforming growth factor-beta in the porcine ovary and its secretion in vitro.

Porcine granulosa cells isolated from small (1-3 mm in diameter) follicles proliferate rapidly in culture in response to 10% fetal bovine serum (FBS) and epidermal growth factor (EGF) (10 ng/ml). Transforming growth factor-beta (TGF beta) inhibits FBS/EGF-stimulated proliferation in a dose-dependent manner. We have used this proliferation inhibitory property of TGF beta to assay qualitatively, the presence of this growth factor in conditioned medium from cultured follicle cells as well as in partially purified preparations from porcine ovarian compartments. In addition, the concentration of TGF beta in the theca cell conditioned medium was quantitatively estimated by generating a TGF beta-dose-response curve (inhibition of FBS/EGF-stimulated proliferation of granulosa cells in monolayer culture) using authentic human TGF beta-1. Ovarian thecal cells isolated from small and large size follicles in the pig ovary secrete TGF beta-like activity in vitro. Medium conditioned by thecal cells in primary monolayer culture contains a latent form of TGF beta which can be activated by heat or acid treatment. In contrast, and unlike rat granulosa cells, porcine granulosa cells in primary monolayer culture do not secrete detectable levels of TGF beta-like activity in the medium. Incubation of heat-activated thecal cell conditioned medium with a TGF beta-neutralizing antibody (which recognizes TGF beta-1 and 2) but not nonimmune serum attenuated the TGF beta-like activity in thecal cell conditioned medium suggesting that this activity is due to authentic TGF beta. Since many cell types secrete latent TGF beta in the medium when cultured in vitro, we next investigated whether thecal cell secretion of latent TGF beta was a function of cell culture or whether the ovarian thecal compartment actually contained detectable levels of TGF beta-like activity. To this end, we used an acid-ethanol extraction procedure to isolate thecal proteins from fresh-frozen tissue. The acid-ethanol extracted protein fraction was mixed with trace amounts of 125I-TGF beta for detection and chromatographed on Bio-Gel P-60 column under acidic conditions. Elution of TGF beta bioactivity from the Bio-Gel P-60 column as measured by inhibition of granulosa cell proliferation correlated with the elution of radioiodinated authentic TGF beta. Preincubation of TGF beta-like activity-containing fractions with TGF beta-neutralizing antibody attenuated the proliferation-inhibitory activity in these fractions. TGF beta activity was also observed in fractions extracted from porcine corpora lutea.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of conspecific males on the oestrous cycle of underfed female mice.

Female mice maintained on restricted diet (3.5, 3.0 and 2.5 gm/female/day) exhibited disturbances in the oestrous cycle which were directly related to the degree of diet restriction. Females maintained on restricted diet, 2.5 gm/day, showed complete suppression of the oestrous cycle. The presence of a conspecific male was effective in maintaining normal oestrous cycles in underfed females to some extent. It is suggested that the ability of males to maintain oestrous cycle in underfed females is due to the stimulatory influence of the male-originating pheromone on hypophysial gonadotrophin secretion.

Studies of the male-originating pheromones involved in the Whitten effect and Bruce effect in mice.

Experiments were designed to elucidate the mode of transmission of the male-originating pheromones involved in the induction of estrus (the Whitten effect) and in implantation failure (the Bruce effect) in mice. The Whitten effect was induced in unisexually grouped females by exposure to corralled males, and also by corralled males housed within a perforated cage (which prevented physical contact of the females with the male-originating pheromone). The results suggest that the pheromone involved in the Whitten effect is volatile (airborne). Implantation failure occurred in a significantly high proportion of newly inseminated females when they were individually confined in corrals and housed below corralled alien males. By contrast, implantation failure was significantly reduced when corralled females were housed above corralled alien males. The results indicate that the male-originating pheromone involved in the Bruce effect is nonvolatile and acts on the females through contact. It is suggested that the pheromone involved in the Whitten effect is distinct from the one involved in the Bruce effect.

Evaluation of the involvement of the adrenals in the pheromonal influences on the oestrous cycle of laboratory mice.

Unisexual grouping of female mice induced irregularities in their oestrous cycles. However, no significant difference in body weight and in the weights of adrenals, uteri and ovaries and in the adrenal delta 5 3 beta-hydroxysteroid dehydrogenase (HSD) activity was found between unisexually grouped females and individually housed females. Adrenalectomy of females failed to prevent the oestrous cycle irregularities induced by unisexual grouping. Furthermore, adrenalectomized females in unisexual groups responded to exposure to males by exhibiting oestrus acceleration and synchronization (the Whitten effect). The results rule out the involvement of the adrenals in the oestrous cycle irregularities induced by unisexual grouping and in the induction of the Whitten effect in unisexually grouped females.

Oestrus induction in unisexually grouped mice by multiple short-term exposures to males.

Oestrus induction and synchronization (the Whitten effect) were achieved in unisexually grouped female mice by short-term (10 and 30 min) exposure to conspecific males.