Antonio Pellicer

Outcome of in vitro fertilization in women with low response to ovarian stimulation

The occurrence of low-response (LR) cycles (defined as peak estradiol levels less than 300 pg/ml) in an in vitro fertilization program using a 3 ampule/day human menopausal gonadotropin regimen were retrospectively reviewed. LR occurred in 51 of 564 patients (9%). The LR serum estradiol levels were categorized into four different patterns that were further analyzed for outcome in these initial cycles, as well as for their predictive value of response in subsequent in vitro fertilization cycles. Changing the stimulation protocol to a combination of clomiphene citrate and human menopausal gonadotropin did not improve the ovarian response in this group of LR patients, and their chance to complete a subsequent normal-response treatment cycle was 32%. Suggestions are made to predict outcome and govern management of women with previous LR cycles.

Intraovarian markers of follicular and oocyte maturation

The use of ovulation induction for multiple follicular growth in in vitro fertilization (IVF) has introduced the problem of follicular asynchrony. As a consequence of the asynchrony, the parameters most commonly used by IVF groups to assess follicular and oocyte quality within those follicles are not sufficiently sensitive or specific. Thus, each follicle must be considered separately, and specific markers of follicular and/or oocyte maturation must be sought from within the follicle. In this review we analyze previous reports of potential markers of follicular and oocyte maturation. In regards to the follicular fluid constituents, the level of estradiol in follicular fluid correlates with fertilization and pregnancy in stimulated cycles. Other steroids are only helpful when specific stimulation protocols are used. The level of some follicular proteins such as alpha-1-antitrypsin and fibrinogen also correlates with fertilization and pregnancy outcome. Cyclic AMP levels in follicular fluid are significantly reduced in follicles leading to conception. Regulators of oocyte maturation, such as the Oocyte Maturation Inhibitor (OMI) or the Meiosis Inducing Substance (MIS) have also been correlated with IVF outcome, but their exact structure remains still unknown. In addition, other sophisticated parameters, such as chemotactic activity of human leukocytes, or simple methods, such as the presence of intrafollicular echoes, have also been used as successful markers in predicting IVF outcome.

Ovarian stimulation for in vitro fertilization and embryo transfer, human menopausal gonadotropin versus pure human follicle stimulating hormone: a randomized prospective study

A randomized, prospective study was conducted to compare ovarian stimulation with human menopausal gonadotropin (hMG) and human follicle-stimulating hormone (hFSH) in an in vitro fertilization and embryo transfer (IVF-ET) program. Minimal inclusion criteria included age less than or equal to 37, tubal infertility, regular menstrual cycles before IVF, and a normal semen analysis. Equivalent doses (225 IU/day) of either hMG (N = 20) or hFSH (N = 20) were administered, and the patients followed by serum estradiol (E2) levels and pelvic ultrasound. Parameters related to the ovarian response to therapy, the number and quality of ova recovered, and the cycle outcome were compared in the two groups using the Students t-test and chi-square analysis. No difference was detected between the groups in peak E2 levels (828 +/- 78 versus 819 +/- 79 in the hMG and hFSH groups, respectively), day of human chorionic gonadotropin (hCG) administration (9.3 +/- 0.3 versus 9.7 +/- 1.01), occurrence of spontaneous luteinizing hormone (LH) surge (44% versus 27%, P greater than 0.05, chi square analysis), average number of ova recovered (5.0 +/- 0.7 versus 5.6 +/- 1), ova maturation (7.5% versus 12.7% rate of immature ova), rate of normal and abnormal fertilization (9.2% versus 8.1% polyspermic fertilization), cleavage stage at transfer (3.6 +/- 0.4 versus 3.4 +/- 0.7 cells per embryos), the number of embryos transferred (2.5 +/- 0.3 versus 2.6 +/- 0.3), or the occurrence of pregnancy (1 in the hMG group and 2 in the hFSH group).(ABSTRACT TRUNCATED AT 250 WORDS)

Manifestation of diabetes mellitus on mouse follicular and pre-embryo development: Effect of hyperglycemia per se

Animal models of diabetes mellitus during pregnancy have repeatedly suggested that maternal hyperglycemia was teratogenic during organogenesis, and thus may contribute to diabetic teratogenesis. However, little attention has been focused on the effects of hyperglycemia on pre-organogenic development. In this report, we examine the effect of hyperglycemia (950 mg glucose/dL) on the development of mouse pre-embryos in vitro. B6C3F1 mice were superovulated with 5 U pregnant mare serum gonadotropin (PMSG) followed by 5 U human chorionic gonadotropin (hCG) 48 hours later. Two cell pre-embryos were recovered 48 hours later, pooled together, and randomly assigned to different treatment groups. Cultures were performed in HAMs F-10 media (Gibco, Long Island, NY) with 0.1% bovine serum albumin (BSA; Sigma, St. Louis, MO) BSA at 37 degrees C in an atmosphere of 5% CO2, 5% O2, and 90% N2 with 15 to 30 embryos per milliliter of culture fluid. Cultures were viewed daily at 24, 48, and 72 hours after culturing, with recording of the development. Compared with control pre-embryos (n = 216), embryos cultured in elevated glucose levels (950 mg/dL) (n = 226) demonstrated marked growth retardation as assessed both by (1) distribution of developmental stages at each observation point (24 hours, P less than .001; 48 hours, P less than .006; 72 hours, P less than .001); and (2) a difference in the average rank sums indicating a delay in maturation (P less than .005). In a second protocol group, pre-embryos were cultured in an equivalent amount of L-glucose; no impairment in development compared with controls was noted.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of periovarian adhesions on follicular development in patients undergoing ovarian stimulation for in vitro fertilization-embryo transfer.

It has been suggested that the presence of periovarian adhesions might impair the ovarian response to gonadotropins. Periovarian adhesions were recorded in 49 women, and the total percentage of accessible ovarian cortex was described at the initiation of the operative procedure. Adhesiolysis was performed as needed for oocyte recovery. Ovarian access did not correlate with serum estradiol level on either the day of human chorionic gonadotropin (hCG) administration or the day after hCG administration. Similarly, neither the total number of follicles on the day of hCG or on the day after hCG, nor the number of follicles 1.0 to 1.4 cm or greater than or equal to 1.5 cm correlated with ovarian access. We conclude that periovarian adhesions are not a major determinant of the ovarian response to gonadotropin stimulation.

Follicular development is impaired by inhibitors of serine proteases in the rat

Serine proteases such as plasminogen activators are produced by granulosa cells both in vivo and in vitro and have been implicated in the process of ovulation. For a study of potential roles of serine proteases in early follicular development, immature rats were injected with pregnant mare serum gonadotropin, followed 2 hours later by laparotomy and injection of the serine protease inhibitors benzamidine and epsilon-aminocaproic acid into the bursa of one ovary. As a control, saline solution was injected into the contralateral bursa. Animals were put to death 48 hours after injection of serine protease inhibitors, and three to five randomly selected longitudinal sections were evaluated by computerized morphometry. The area occupied by antral follicles relative to the total cross-sectional area of each section was computed. Resultant ratios from serine protease inhibitor-treated ovaries were compared with those from contralateral control ovaries. Ninety-three percent of serine protease inhibitor-treated ovaries showed a reduction in antral follicular size when compared with corresponding control ovaries, which is indicative of inhibitory effects of serine protease inhibitor treatment on folliculogenesis. To further investigate this effect, ovulation was induced by human chorionic gonadotropin administration 48 hours after pregnant mare serum gonadotropin and 46 hours after serine protease inhibitor or saline solution treatment. Animals were put to death 20 hours later and the number of oocytes ovulated into oviducts was determined. Oviducts from serine protease inhibitor-treated ovaries contained 51% fewer oocytes than their control counterparts. Artifacts of surgical stress or vascular diffusion of serine protease inhibitor from treated to control sides were ruled out by appropriate control experiments. We conclude that early serine protease inhibitor treatment of pregnant mare serum gonadotropin-stimulated rat ovaries impairs folliculogenesis. Thus, in addition to involvement in ovulation, serine proteases appear to play important roles throughout follicular development.

The effect of the incubation temperature on the cleavage rate of mouse embryos in vitro

We have studied the effect of an elevated incubation temperature on the in vitro cleavage rate of one- and two-cell mouse embryos. Two-cell embryos demonstrated a significantly higher rate of cleavage when incubated at 39°C as compared to 37 or 41°C. When recovered at the onecell stage, the difference in cleavage rate between the groups incubated at 37 and 39°C did not reach significance. While the morphology of the embryos incubated at 37°C did not differ from that of the embryos incubated at 39°C in either group, a significantly higher rate of degeneration was noted in the group of two-cell embryos incubated at 41°C. These findings may apply to the human in vitro fertilization and embryo transfer system (IVF-ET), where the existing “lag” between embryo and endometrium could be narrowed if embryo cleavage rates could be accelerated. Further documentation of the normality of these “advanced” embryos is required.

Desensitization to follicle-stimulating hormone in cumulus cells is coincident with hormone induction of oocyte maturation in the rat follicle

The objective of the present studies was to assess whether hormone induction of oocyte maturation in isolated intact follicles may be linked to desensitization of follicle-stimulating hormone (FSH) in the oocyte-cumulus complex (OCC). Incubation of follicles with chorionic gonadotropin (hCG), FSH or epidermal growth factor (EGF) produced a marked inhibition of FSH-dependent cyclic AMP accumulation in OCC with a time-course coincident with the onset of germinal vesicle breakdown (GVBD). These effects were evident within 3 h for both hCG and FSH, but with EGF a reduced response to FSH was seen within 1 h of treatment followed by an increase in GVBD. In contrast, no inhibition of cyclic AMP accumulation was seen in response to cholera toxin, forskolin or LH in OCC derived from follicles incubated with hCG for 3 h. The time-course for induction of oocyte maturation by incubation of the intact follicle with hCG was also coincident with production of prostaglandin (PG) F2 alpha, an indirect marker of cyclooxygenase induction. No effect on metabolic coupling between the oocyte and cumulus cells was seen until 9 h after hCG treatment. Retinoic acid caused a marked decrease in metabolic coupling between the oocyte and cumulus cells but inhibited oocyte maturation both in denuded oocytes and OCC. Since FSH desensitization in OCC, the resumption of meiosis, and production of arachidonic acid-derived products were coincident, it is suggested that abrogation of FSH action in cumulus cells by the ovulatory surge of gonadotropins may initiate oocyte maturation.

Improved Oocyte Quality through Improved Ovulation Induction Regimen

The key to an improved success rate in in vitro fertilization and embryo transfer (IVFET) lies either in improving implantation rates or in improving embryo quality. The best way to improve embryo quality, at least at our current level of understanding, is to improve oocyte quality. The theoretical question posed then is: Can oocyte quality be improved by better means of superovulation protocols? The process of multiple follicular development using superovulatory agents should be analyzed and new strategies developed on the basis of the physiological mechanism of selection and follicular development in spontaneous cycles.

Spironolactone does not affect in vitro androgen secretion by human granulosa-luteal cells.

Although spironolactone has been documented to decrease peripheral serum androgen levels in women treated for androgen excess of ovarian origin, the site of action does not appear to be the granulosa cell. In vitro cell culture studies using human granulosa-luteal cells isolated from women undergoing ovarian stimulation for IVF have shown no affect on T, delta 4A, or DHT secretion in the presence of clinically therapeutic levels of spironolactone (0-10(-6) M). In addition, neither P nor E2 production was affected by increasing concentrations of spironolactone.