Reproductive biology and IVF: ovarian stimulation and endometrial receptivity.

The influence of ovarian stimulation on endometrium receptivity has been inadequately addressed in medical literature. Hormonal effects of ovarian stimulation on endometrial changes as compared with the natural cycle should be elucidated and correlated with the potential of the embryo to implant. It is important to distinguish between the endometrial effect of induction of ovulation in anovulatory women and those of ovarian (super)ovulation in ovulatory women. Induction of ovulation leads to in vivo conception whereas ovarian stimulation results in in vitro fertilization. The available data in the field indicate that endometrial changes have an impressive negative influence on the potential of embryonic implantation. The aim of this review is to analyse the effects of gonadotropin, GnRH-agonist and GnRH-antagonist administration on endometrial behaviour, to highlight the gaps in current knowledge and to propose areas in which research is needed.     

Progress in ovarian stimulation.

It is uncertain whether patients who do not ovulate but fail to conceive following CC therapy behave differently during gonadotropin therapy. However, if the infertile patient with PCOS is resistant to anti-estrogen therapy, gonatropin treatment administered through low dose protocols should be considered. As compared to conventional regimens, complication rates are reduced despite similar efficacy. It should be recognized that patients with PCOS constitute a very heterogeneous group. Therapy outcome may benefit from improved classification. The identification of clinical characteristics that identify those women with anovulation who are unlikely to respond to clomiphene would permit earlier use of gonadotropin therapy and potentially offer major health and economic benefits. Furthermore, if ovarian responsiveness to ovulation-induction therapy could be predicted for an individual patient, it might be possible to devise regimens that reduced the risk of ovarian hyperstimulation and multiple pregnancy.     

Follicular and luteal function in the puberal rat: gonadotrophic hormone stimulation and enzymic-nucleotide interactions.

Using the puberal rat and the PMS-treated rat as animal systems, ovarian events associated with follicular and luteal development have been characterized by measuring gonadotrophic hormone (LH, FSH and prolactin) and progesterone concentrations in peripheral serum; and selected enzymic (NAD-kinase:NAD-K and glucose-6-phosphate dehydrogenase: G6PD) activities and nucleotide (NAD, NADH, NADP, NADPH, ATP) concentrations in ovarian tissue. In the puberal rat, the period of follicular development was characterized by increased ovarian NAD-K SA, NAD and NADH concentrations and decreased ATP and NADP concentrations. The first pro-oestrus was characterized by greatly elevated LH, FSH, prolactin and progesterone concentrations, significant decreases in ovarian NAD-K SA, NAD, NADP and ATP concentrations, and an increase in NADPH concentrations. The development of new corpora lutea was associated with striking increases in ovarian NAD-K SA and G6PD SA. Increased activity of both enzymes exhibited a significant positive coefficient of correlation with the number of corpora lutea contained within the ovarian tissue. PMS (4 IU) stimulation of follicular activity resulted in events leading to the induction of an endogenous LH surge and ovulation. Associated with increased follicular activity was increased ovarian NAD-K SA. In contrast to the puberal rat, no rise in progesterone concentrations was associated with the LH surge or the formation of corpora lutea.     

Myocardial infarction complicating the initial phase of an ovarian stimulation protocol

Two previous reports have reported myocardial infarction during ovarian hyperstimulation syndrome, a complication of controlled ovarian stimulation characterized by ascites, pleural effusion, hemoconcentration and an increased thromboembolic risk, but no association with the initial phase (before treatment with human chorionic gonadotropin) of a normal ovarian stimulation protocol for infertility has ever been described. We report the first case, to our knowledge, of acute myocardial infarction occurring during the initial phase of an otherwise uncomplicated ovarian stimulation protocol. A young woman with infertility associated to polycystic ovary syndrome was treated with leuprolide acetate and recombinant follicle stimulating hormone to induce ovarian stimulation for in vitro fertilization and embryo transfer. After 12 days the patient presented a non-ST elevation myocardial infarction, which was treated with aspirin, clopidogrel, enoxaparin, intravenous nitrates and beta blockers. Cardiac catheterization showed angiographically normal coronary arteries. Echocardiography showed a circumscribed akinesis of the inferior apical segment of the left ventricle and right ventricular apex, which was confirmed by cardiac magnetic resonance. A screening for thrombophilic diathesis was negative. The patient was discharged and remained asymptomatic at 1 and 3 months follow up. Further ovarian stimulations were excluded and a trial of oocyte retrieval on spontaneous cycle was planned. Myocardial infarction can complicate ovarian stimulation protocols for infertility even in their early phase without any sign of ovarian hyperstimulation syndrome.     

Receptor regulation and target cell responses: Studies in the ovarian luteal cell

Studies of hormone-induced ‘desensitization’ in the luteinized rat ovary show that changes in receptor number and adenylate cyclase at the cell surface result in an altered biological effect of luteinizing hormone on progesterone production. The sensitivity of these effects to changes in gonadotropin levels suggests that receptor turnover or processing is involved in the normal mechanism of hormone action.     

The effect of luteal phase estrogen antagonism on endometrial development and luteal function in women

Previous studies of the role of estrogen in primate luteolysis, designed to investigate the effects of estrogen antagonism or selective inhibition of luteal phase estrogen production on luteal function, have ignored the impact of such treatments on secretory endometrial development. We examined the effect of luteal phase estrogen antagonism on endometrial maturation and luteal function in six women. In each of two menstrual cycles in each woman, blood samples were obtained on alternate days from cycle days 3-9, daily until 1 day after the urinary LH surge (day 0), and again on alternate days until the onset of menses. In the second of each pair of cycles, clomiphene citrate (100 mg) was administered daily from 2 days after the LH surge until menses. Endometrial biopsy was performed 13 days after the LH surge in each cycle. Serum FSH, LH, estradiol, and progesterone (P) were measured by RIA. The endometrial histological date and concentration of cytosolic (C) and nuclear (N) estrogen (ER) and P (PR) receptors were determined. We found significant (P less than 0.05) increases in luteal phase serum FSH, LH, estradiol, and P levels in the clomiphene cycle compared to those in the control cycle. Endometrial histology was significantly (P less than 0.002) different during estrogen antagonism; a maturation delay of more than 2 days was found in all six women during the clomiphene cycle. Luteal phase duration was unchanged by clomiphene (P = 0.29). Endometrial ER-C [7.38 +/- 2.52 (+/- SEM) vs. 38.75 +/- 10.17 fmol/mg protein], ER-N (248 +/- 84 vs. 685 +/- 80 fmol/mg DNA), and PR-C (97 +/- 38 vs. 189 +/- 38 fmol/mg protein) were significantly lower (P less than 0.03) in the clomiphene cycle than in the control cycle, whereas PR-N was not different (P greater than 0.10). These data suggest that luteal phase estrogen 1) modulates endometrial PR and 2) plays an important role in secretory endometrial development.     

Gonadotropic stimulation of inhibin secretion by the human ovary during the follicular and early luteal phase of the cycle

We studied the pattern of secretion of inhibin bioactivity from the ovary into peripheral blood during the follicular and early luteal phase of the menstrual cycle in women receiving gonadotropin therapy. Multiple follicular development was stimulated in 5 women undergoing in vitro fertilization and embryo transfer for tubal infertility using three different treatments designed to vary the concentration of FSH and LH (14 cycles). The women received clomiphene citrate (150 mg/day) from days 2-6 alone or supplemented with either exogenous human menopausal gonadotropin (28 IU/3 h) or pure FSH (28 IU/3 h) from day 6 until the day of follicle aspiration. Inhibin concentrations increased 10-fold in parallel with those of estradiol, from 0.2-0.3 U/mL on day 2 (before the onset of treatment) to 4-5 U/mL on day 14 of the cycle (time of the peak LH level). Coincidental to the LH surge, the inhibin concentration declined 2- to 3-fold before increasing again early in the luteal phase. The concentration of inhibin was higher in the gonadotropin-treated group (clomiphene plus human menopausal gonadotropin/FSH) than in the group treated with only clomiphene during the follicular phase. The number of follicles stimulated was significantly higher (P less than 0.001) in the group given exogenous gonadotropins [4.8 +/- 0.4 (SE)] than in the clomiphene alone group (2.2 +/- 0.4). These data strongly suggest that both the Graafian follicles and the corpus luteum secrete inhibin, which together with estradiol and progesterone may play a role in the regulation of FSH secretion during the luteal phase.     

Vascular endothelial growth factor Trap suppresses ovarian function at all stages of the luteal phase in the macaque

CONTEXT: Fertility is dependent on a functioning corpus luteum, the formation of which is associated with intense angiogenesis. The role of angiogenic factors, such as vascular endothelial growth factor (VEGF), in luteal function has yet to be defined in primates. OBJECTIVE: The objective of this study was to determine effects of inhibiting VEGF by a VEGF Trap, a receptor-based inhibitor, administered at the early or midluteal phase, on pituitary-ovarian function. DESIGN: Effects of a single injection of VEGF Trap at three doses in the early luteal phase or a single dose in the midluteal phase were investigated and compared with control cycles. SETTING: This work was conducted in the Primate Unit. PARTICIPANTS: Eleven stump-tailed macaques with regular ovulatory cycles participated in this study. VEGF Trap was well tolerated, and all completed the study. INTERVENTIONS: A single injection of VEGF Trap at a dose of 4, 1, or 0.25 mg/kg was administered in the early luteal phase or 1 mg/kg in the midluteal phase. Controls received vehicle or the constant region of human IgG. MAIN OUTCOME MEASURES: Changes in serum concentrations of progesterone, estradiol, LH, FSH, inhibin A, VEGF Trap, and menstrual bleeding were the main outcome measures. RESULTS: Early luteal treatment caused a significant attenuation of the normal serum progesterone and estradiol concentrations, followed by a marked increase in LH and FSH. Inhibin A was not significantly reduced. After 1- and 4-mg/kg doses, progesterone remained suppressed throughout the luteal phase, and premature menstruation occurred; whereas the response to the 0.25-mg/kg dose was transitory, and menstruation was at the normal time. Midluteal treatment also resulted in a significant suppression of progesterone secretion. CONCLUSIONS: VEGF is essential for both the development and maintenance of luteal function.     

Luteolysis-induced changes in phase composition and fluidity of bovine luteal cell membranes.

X-ray diffraction, fluorescence polarization of trans-parinaric acid, and fluorescence photobleaching recovery of dioctadecyltrimethyneindolecarbocyanine have been used to characterize the phase composition and liquid phase fluidity of bovine luteal cell membranes and membrane lipids for functional corpora lutea collected at midcycle and for regressing corpora lutea collected after treatment with prostaglandin F2 alpha. These results support previous observations of gel phases in microsomal preparations of regressed luteal cells at physiological temperatures and further suggest that the plasma membrane may be the main source of this gel phase. Analysis of the overall lipid composition of the microsomal preparations from these cells indicates a role for sphingomyelin, in the presence of cholesterol, for the generation of a gel phase at physiological temperatures.     

The effect of luteal phase estrogen antagonism on luteinizing hormone pulsatility and luteal function in women

Pulsatile LH secretion was studied to determine if the frequency of LH pulses was altered by the administration of clomiphene citrate (CC; 150 mg) for 5 days during the midluteal phase of the menstrual cycle. Seven normal women received CC or placebo in alternate cycles in a randomized double blind fashion. On the day after drug administration, blood samples were obtained at 15-min intervals for 8 h for serum LH determinations. Daily blood samples were also obtained throughout the luteal phase for determination of serum LH, estradiol (E2), and progesterone. LH pulse frequency increased from 2.4 +/- 0.5 (+/- SEM)/8 h after placebo to 3.9 +/- 0.6/8 h (P less than 0.01) after CC treatment, but pulse amplitude did not change. The transverse mean of serum LH was higher after CC (13.6 +/- 0.5 mIU/ml) than after placebo (8.4 +/- 0.3 mIU/ml; P less than 0.001), and luteal phase length was increased from 13.5 +/- 0.5 to 16.0 +/- 0.4 days (P less than 0.001) by administration of CC. Luteal phase levels of E2 and progesterone measured daily were significantly elevated (P less than 0.01) in CC-treated cycles. These findings suggest that CC increases the frequency of hypothalamic GnRH secretory episodes, perhaps by an action involving a decrease in endogenous opioid peptide activity. Since peripheral progesterone levels were elevated in the CC-treated cycles, E2 may play a permissive role in the ability of progesterone to increase endogenous opioid peptide activity acutely. Furthermore, since the luteal phase was significantly prolonged by an increase in endogenous LH pulse frequency, the slow frequency of LH pulses in the normal late luteal phase may contribute to the onset of luteolysis in the human.     

Progesterone, androgen and estradiol production by porcine luteal cell subpopulations: dependence on cell composition and periods of luteal phase

The size and number small (Sc) and large (Lc) luteal cells and the steroids they secrete were determined in porcine corpora lutea (CL) collected on days 1-3, 8-10 and 14-16 of the estrus cycle. The cells were separated with the aid of Ficoll linear gradient. The size of Sc increased in middle-luteal phase (MLP) in comparison with early luteal phase (ELP) and retained consistent value until the end of the luteal phase (LLP). Lc increased in size with advancing luteal phase, their number per CL being greater in MLP than in ELP but decreased in LLP. In contrast, the number of Sc decreased in MLP in comparison with ELP and did not change in LLP. At the initial luteal phase the majority of small cells did not show any histochemical reactivity for delta5-3?-hydroxysteroid dehydrogenase which then increased with luteal phase progress. Lc collected during ELP showed 5.7 times higher androgen secretion than Sc. A decrease of androgen secretion by Lc was observed as the luteal phase progressed but it was three times higher in Sc than in Lc collected during LLP. Both types of luteal cells produced estradiol even without the addition of substrates. Endogenous estradiol content was always higher in Lc than in Sc. Moreover, estradiol secretion by both cell types was higher in LLP than in MLP and ELP. These findings suggest that the view on granulosa origin of Lc and theca origin of Sc as raised by others may be oversimplified. In appears that Sc most likely undergo hypertrophy, but retain their prior phenotype and also that some Lc gradually dedifferentiate to Sc as the corpus luteum ages.     

An integrated view on the luteal phase: diagnosis and treatment in subfertility

The term ‘luteal phase deficiency’ was first coined more than 60 years ago, and, since then, it has been suggested as a clinical entity per se and an aetiological factor for subfertility, implantation failure and recurrent miscarriage. Despite the existing recommendations for rational work‐up in subfertility, luteal phase evaluation and progesterone therapy alone is still common in daily practice. This review comprises results from a Pubmed literature search with the terms ‘luteal phase’ and ‘subfertility’, focussing on clinical situations not primarily related to assisted reproduction techniques. Additional data from the experimental studies published in the past 10 years on follicular maturation, oocyte developmental competence and the ovulatory cascade are integrated into the clinical continuum of dysfunctional ovulation, menstrual cycle irregularity and impaired corpus luteum function. As reliable diagnostic tools for adequate luteal function are missing, the presence of clinical symptoms such as cycle irregularity or premenstrual spotting is indicative and should initiate early follicular phase diagnostic work‐up. New evidence on the interdependence of oocyte and follicular maturation and resulting developmental competence of the embryo further support the use of ovarian stimulation as the first‐line therapeutic option in different subsets of patients with subfertility including luteal phase deficiency.     

Longitudinal evaluation of the luteal phase and its transition into the follicular phase

The precise patterns of LH, FSH, and PRL secretion and their correlation with estradiol (E2) and progesterone (P) secretion during the entire luteal phase have not been elucidated. To analyze in detail the secretory patterns of these hormones we performed 29 consecutive studies in 5 healthy, regularly menstruating women throughout their luteal phase [days 0 (ovulation), 2, 6, 10, and 14] and subsequent early follicular phase (day 2F). During each study plasma LH, FSH, PRL, E2, and P were measured at 10-min intervals for 6 h. Both plasma LH concentrations and LH pulse frequency declined from days 0 to 10 and increased thereafter, whereas LH pulse amplitude continued to decline throughout the luteal and early follicular phases. Plasma FSH concentrations followed a pattern similar to that of LH; however, there was a larger increase in the FSH level on days 14 and 2F. Plasma PRL levels declined initially on day 2 and again on day 14. Regression analysis indicated a positive correlation between LH concentrations and LH pulse frequency (r = 0.715; P less than 0.001) and between PRL and E2 concentrations (r = 0.528; P less than 0.01). A negative correlation was found between plasma P concentrations and both LH concentrations (r = -0.521; P less than 0.01) and LH pulse frequency (r = -0.633; P less than 0.001) and between plasma E2 and FSH concentrations (r = -0.762; P less than 0.001). Thirty-six (65%) PRL pulses and only 11 (39%) FSH pulses coincided with LH pulses. There was no clear pulsatile pattern of secretion of either E2 or P. We conclude that 1) the plasma LH, FSH, PRL, E2, and P concentrations vary markedly throughout the luteal phase; 2) the plasma LH level is largely dependent on the frequency of LH pulses; 3) plasma P decreases plasma LH by reducing the frequency of LH pulses; 4) the remarkable synchrony between PRL pulses and LH pulses implies that their secretion may be regulated by a common neuroendocrine mechanism; and 5) the preferential increase in FSH during the late luteal phase may play an important role in follicular recruitment for the subsequent cycle.