GnRH agonist and hCG (dual trigger) versus hCG trigger for final follicular maturation: a double-blinded, randomized controlled study.

STUDY QUESTION: Does co-administration of GnRH agonist and Human chorionic gonadotropin (hCG; dual trigger) in IVF cycles improve the number of mature oocytes and pregnancy outcome compared to hCG alone? SUMMARY ANSWER: Using the dual trigger for final follicular maturation increases the number of oocytes, mature oocytes and number of blastocysts (total and top-quality) compared to triggering with hCG alone. WHAT IS KNOWN ALREADY: hCG is used at the end of controlled ovarian hyperstimulation as a surrogate LH surge to induce final oocyte maturation. Recently, based on retrospective studies, the co-administration of GnRH agonist and hCG for final oocyte maturation (dual trigger) has been suggested to improve IVF outcome and pregnancy rates. STUDY DESIGN, SIZE, DURATION: A single center, randomized controlled, double-blinded clinical trial between May 2016 and June 2018 analyzed by intention to treat (ITT). PARTICIPANTS/MATERIALS, SETTINGS, METHODS: One hundred and fifty-five normal responder patients were randomized either to receive hCG or dual trigger for final oocyte maturation. Data on patients age, BMI, AMH, number of oocytes retrieved, number of metaphase 2 (MII) oocytes, zygotes and blastocysts, clinical pregnancy rate and live birth rate were assessed and compared between the dual trigger group and the hCG group. We performed a planned interim analysis after the recruitment of 50% of the patients. Based on the totality of outcomes at the interim analysis we decided to discontinue further recruitment. MAIN RESULTS AND THE ROLE OF CHANCE: One hundred and fifty-five patients were included in the study. The age (36 years versus 35.3 years P = NS), BMI (24 kg/m2 versus 23.7 kg/m2) and the AMH (20.1 pmol/l versus 22.4 pmol/l) were comparable between the two groups. Based on ITT analysis, the number of eggs retrieved (11.1 versus 13.4, P = 0.002), the MII oocytes (8.6 versus 10.3, P = 0.009), total number of blastocysts (2.9 versus 3.9, P = 0.01) and top-quality blastocysts transferred (44.7% versus 64.9%; P = 0.003) were significantly higher in the dual trigger group compared to the hCG group. The clinical pregnancy rate (24.3% versus 46.1%, OR 2.65 (1.43-1.93), P = 0.009) and the live birth rate per transfer (22% versus 36.2%, OR= 1.98 (1.05-3.75), P = 0.03) were significantly higher in the dual trigger group compared to the hCG group. LIMITATIONS, REASONS FOR CAUTION: None. WIDER IMPLICATIONS OF THE FINDINGS: The enhanced response observed with the dual trigger might lead to better IVF outcomes were it used more widely. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by TRIO Fertility. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov identifier: NCT02703584. DATE OF TRIAL REGISTRATION: March 2016. DATE OF FIRST PATIENT'S ENROLLMENT: May 2016.

Aromatic hydrocarbon receptor-driven Bax gene expression is required for premature ovarian failure caused by biohazardous environmental chemicals.

Polycyclic aromatic hydrocarbons (PAHs) are toxic chemicals released into the environment by fossil fuel combustion. Moreover, a primary route of human exposure to PAHs is tobacco smoke. Oocyte destruction and ovarian failure occur in PAH-treated mice, and cigarette smoking causes early menopause in women. In many cells, PAHs activate the aromatic hydrocarbon receptor (Ahr), a member of the Per-Arnt-Sim family of transcription factors. The Ahr is also activated by dioxin, one of the most intensively studied environmental contaminants. Here we show that an exposure of mice to PAHs induces the expression of Bax in oocytes, followed by apoptosis. Ovarian damage caused by PAHs is prevented by Ahr or Bax inactivation. Oocytes microinjected with a Bax promoter-reporter construct show Ahr-dependent transcriptional activation after PAH, but not dioxin, treatment, consistent with findings that dioxin is not cytotoxic to oocytes. This difference in the action of PAHs versus dioxin is conveyed by a single base pair flanking each Ahr response element in the Bax promoter. Oocytes in human ovarian biopsies grafted into immunodeficient mice also accumulate Bax and undergo apoptosis after PAH exposure in vivo. Thus, Ahr-driven Bax transcription is a novel and evolutionarily conserved cell-death signaling pathway responsible for environmental toxicant-induced ovarian failure.

Induction of luteolysis in the human with a long-acting analog of luteinizing hormone-releasing factor.

Subcutaneous injection of 50 micrograms of a long-acting analog of luteinizing hormone-releasing factor on each of two successive days during mid-luteal phase in normally cycling women induced a short luteal phase and premature menstruation. These events were associated with luteolysis, as evidenced by the consistent and parallel premature decline of progesterone and estradiol levels compared with those in control cycles. This finding may prove to be useful in the prevention or interception of implantation.

Menopausal flushes: a neuroendocrine link with pulsatile luteninizing hormone secreation.

Menopausal flush episodes were found to be invariably associated with the initiation of pulsatile pituitary release of luteinizing hormone. This was not accompanied by a significant change in circulating catecholamine or prolactin concentrations. Since pulsatile luteinizing hormone release results from episodic secretion of luteinizing hormone releasing factor by the hypothalamus, these findings suggest a link between the neuroendocrine mechanisms that initiate such episodic secretion and those responsible for the onset of flush episodes.

Luteal phase defects induced by an agonist of luteinizing hormone-releasing factor: a model for fertility control.

Subcutaneous injection of 50 micrograms of a luteinizing hormone-releasing factor agonist (LRF agonist) for three successive days at the time of menstruation in normal cycling women induces a shortened luteal phase with suboptimal concentrations of circulating estradiol and progesterone. This luteal phase defect follows a reduced concentration of follicle-stimulating hormone during the follicular phase and a resulting inadequate follicular maturation. Since a short luteal phase is associated with an endometrium not conductive to implantation, administration of the LRF agonist at the onset of menstrual cycle may prove to be a practical and novel approach to fertility control.

Genetic variance modifies apoptosis susceptibility in mature oocytes via alterations in DNA repair capacity and mitochondrial ultrastructure.

Although the identification of specific genes that regulate apoptosis has been a topic of intense study, little is known of the role that background genetic variance plays in modulating cell death. Using germ cells from inbred mouse strains, we found that apoptosis in mature (metaphase II) oocytes is affected by genetic background through at least two different mechanisms. The first, manifested in AKR/J mice, results in genomic instability. This is reflected by numerous DNA double-strand breaks in freshly isolated oocytes, causing a high apoptosis susceptibility and impaired embryonic development following fertilization. Microinjection of Rad51 reduces DNA damage, suppresses apoptosis and improves embryonic development. The second, manifested in FVB mice, results in dramatic dimorphisms in mitochondrial ultrastructure. This is correlated with cytochrome c release and a high apoptosis susceptibility, the latter of which is suppressed by pyruvate treatment, Smac/DIABLO deficiency, or microinjection of 'normal' mitochondria. Therefore, background genetic variance can profoundly affect apoptosis in female germ cells by disrupting both genomic DNA and mitochondrial integrity.

Enhanced endocytotic and transcytotic activity in the rat endometrium prior to embryo implantation.

BACKGROUND: Understanding the mechanisms by which fluid absorption and secretion occur in the endometrium is clinically important since conditions that deregulate this process reduce fertility. It has been suggested that luminal epithelial cells induce a crucial step in the process of embryo implantation called uterine closure via endocytotic fluid uptake. Uterine lumen closure is a key step in the process of embryo implantation and is absent in some infertile strains of mice. METHODS: To investigate the process of uterine closure a ferritin-based tracer, used as a marker of endocytosis, was injected into the uterine lumen on day 5 of pregnancy when closure occurs. RESULTS: Unexpectedly, luminal epithelial uptake of tracer was minimal on day 5 of pregnancy discrediting endocytosis as the induction method of uterine closure. In contrast, ferritin was found deep in the stromal portion of the endometrium in pre-pregnant animals. CONCLUSIONS: We have shown for the first time that uterine closure is not induced by luminal epithelial cell driven endocytosis. Another novel finding of this study was the passage of the tracer ferritin up to 15 cells deep into the endometrium suggesting an as yet unstudied mechanism by which information can be transported from the uterine lumen to the underlying stroma.

Inhibition of osteoclast differentiation by polycyclic aryl hydrocarbons is dependent on cell density and RANKL concentration.

We investigated the effect of representative polycyclic aryl hydrocarbons (PAHs), benzo[a]pyrene (BaP), and 7,12-dimethylbenz[a]anthracene (DMBA) on osteoclast differentiation and function by using dispersed cancellous bone derived rabbit osteoclasts and the RAW264.7 cells. These cells differentiate into osteoclasts when exposed to receptor activator of NF-kappaB ligand (RANKL). The rabbit osteoclasts were exposed to 10(-6) to 10(-9)M BaP or DMBA and the tartrate-resistant acid phosphatase (TRAP)-positive cells were counted. The effect of PAHs on osteoclast differentiation in dispersed rabbit osteoclast-containing stromal cell populations was cell density dependent, suggesting that the cell density of stromal cells, osteoclast precursors, and/or mature osteoclasts are factors regulating the effect of PAHs. To investigate the direct effect of BaP on osteoclast differentiation, RAW264.7 cells were exposed to 10(-5) to 10(-6) M BaP. Treatment of RAW264.7 cells cultured with 25 ng/ml soluble RANKL and 10(-5)M BaP for 5 days decreased osteoclast differentiation, TRAP activity levels, and resorption of bone-like substrata. The inhibition was prevented by 10(-6) to 10(-7) M resveratrol, an aryl hydrocarbon receptor (AhR) antagonist, and by higher concentrations of RANKL. To investigate the ability of RANKL to reverse BaP-mediated inhibition, gene expression was determined by RT-PCR. Cytochrome P450 1B1 (CYP1B1) mRNA, one of the genes activated by BaP, was present only in the groups exposed to BaP; the levels of CYP1B1 mRNA decreased in the presence of increasing concentrations of RANKL. These results suggest that the inhibitory effects of PAHs on osteoclastogenesis are direct and likely involve interaction of the RANKL and PAH signaling pathways.

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.

Menopausal flushes: effect of pituitary gonadotropin desensitization by a potent luteinizing hormone- releasing factor agonist.

Recent evidence suggests that the menopausal flush is linked to the neuroendocrine events which govern pulsatile LH secretion and thermoregulation. This study was designed to determine whether abolishment of LH pulses may abate flush episodes. After pituitary gonadotropin densensitization by a LRF agonist, LH and FSH pulses were abolished, and serum gonadotropin levels were decreased. Flush episodes, however, were unaltered. These findings demonstrate that pulsatile LH release by the pituitary is not causally related to menopausal flushes, and support the contention that flush episodes are initiated by a hypothalamic mechanism(s).

Simultaneous pulsatile release of prolactin and luteinizing hormone induced by luteinizing hormone-releasing factor agonist.

Subcutaneous injection of 50 microgram long acting agonist of LRF (LRF-Ag) resulted in the prompt, simultaneous pulsatile release of both LH and PRL in four hypogonadal women. After repeated daily administration of LRF-Ag. LH release in response to LRF-Ag was markedly attenuated, and LH pulses were abolished. LRF-Ag induced PRL release was also attenuated but remained pulsatile. These findings demonstrate the dissociation of the LRF-Ag-induced contemporaneous release of LH and PRL and suggest a hypothalamic site of action of LRF-Ag on PRL release.

Progestins increase endogenous opioid peptide activity in postmenopausal women.

To determine the effect of administration of a progestin alone on endogenous opioid peptide activity, we infused naloxone (2 mg/h for 4 h) into seven estrogen-deficient postmenopausal women before and after oral medroxyprogesterone acetate (Provera; 20 mg) daily for 30 days. Baseline serum LH levels were significantly decreased by the Provera therapy [70.3 +/- 6.6 (+/- SE) vs. 27.5 +/- 1.7 mIU/ml; P less than 0.001]. Naloxone infusion before Provera treatment had no effect on serum LH levels. In contrast, after Provera therapy, a significant (P less than 0.001) increase in LH levels toward the pre-Provera baseline occurred with naloxone infusion. These findings suggest that progestins exert their negative feedback effects on LH at least in part through an opioid peptide-mediated mechanism and that progestin treatment alone can reestablish opiatergic control of LH.

Prolonged elevation of hypothalamic opioid peptide activity in women taking oral contraceptives.

To examine the hypothesis that endogenous opioid peptide activity is chronically elevated by oral contraceptives, we infused either naloxone or saline into 10 women during the use of, or 5-6 and 9-10 days after stopping, combination birth control pills. A paradoxical increase in prolactin occurred with naloxone infusion during and 5-6 days after stopping the pills. Serum LH levels were not significantly elevated by naloxone until 9-10 days after cessation of pill use. These results suggest that hypothalamic opioid peptide activity is continuously elevated in women taking oral contraceptives.

Thyrotropin and prolactin responses to thyrotropin-releasing hormone in premenstrual syndrome.

Recent reports of altered TSH responsiveness to its releasing hormone (TRH) in women with premenstrual syndrome (PMS) suggested that subclinical hypothyroidism may be responsible for the mood changes, such as depression, that occur in these women. In this study we measured basal and TRH-stimulated serum TSH and PRL levels in 15 women with PMS and in 19 age-matched normal women. The mean baseline serum TSH concentrations were similar in the 2 groups in both the follicular [normal, 1.3 +/- 0.2 (+/- SE); PMS, 0.9 +/- 0.2 mU/L] and luteal (normal, 1.1 +/- 0.2; PMS, 1.1 +/- 0.2 mU/L) phases of the cycle. The mean baseline serum PRL levels also were similar in the 2 groups in the follicular (normal, 16 +/- 2; PMS, 13 +/- 2 micrograms/L) and luteal (normal, 13 +/- 2; PMS, 14 +/- 2 micrograms/L) phases of the cycle. After TRH administration, peak serum PRL and TSH levels were reached at 15 and 30 min, respectively, and the response curves were virtually identical in the 2 groups in both phases of the cycle. One normal woman had elevated basal and TRH-stimulated TSH concentrations compatible with subclinical hypothyroidism, but had normal noncyclic scores on her prospective rating scales. Our findings suggest that PMS is not associated with thyroid dysfunction or abnormal PRL secretion and that thyroid hormone replacement therapy is not indicated in this condition.

Gonadotropin-estradiol responses to a superactive luteinizing hormone-releasing hormone agonist in women.

After the sc administration of 1, 10, and 50 micrograms of the LRF agonist [D-Trp6,Pro9,NEt]LRF, dose-dependent increments in circulating levels of LH, FSH, and estradiol were observed which were 2- to 3-fold greater in the late than in the early follicular phase. The 10-microgram dose of LFR agonist appears to induce a maximal acute gonadotropin-estradiol response. Both 10- and 50-microgram doses of the agonist elicited gonadotropin increments which were several times greater than that seen during the midcycle surge. The only difference between the two doses was the more sustained action on gonadotropin release of the latter. It is estimated that this superactive LRF agonist is approximately 140 times more potent than the decapeptide LRF. These observations provide information useful in the application of this LRF agonist for clinical studies.

Stimulatory effect of 2-hydroxyestradiol on prolactin release in hypogonadal women.

Administration of an intravenous bolus (1.0 mg) or a constant rate infusion (250 microgram/h for 4 h) of 2-hydroxyestradiol (20H-E2) to hypogonadal women, resulted in no discernible alteration in the circulating levels of LH and FSH. In contrast, progressive increments in the release of prolactin were unequivocally detected at 4.5 h after receipt of the bolus and 2.5 h following the initiation of the infusion.

Use of gonadotropin-releasing hormone agonist to trigger follicular maturation for in vitro fertilization.

In spontaneous cycles both LH and FSH are secreted in a surge at midcycle. In in vitro fertilization (IVF) cycles, hCG administration results in elevation of LH-like activity only. The objective of this study was to compare the effectiveness of a single midcycle dose of GnRH agonist with hCG on follicular maturation. Eighteen IVF cycles in 14 women were randomized to receive either 0.5 mg leuprolide acetate or 5000 IU hCG at midcycle. Both groups underwent identical ovarian stimulation and cycle monitoring. On the day of GnRH agonist or hCG administration, estradiol concentrations and the number of follicles 1.5 cm or larger were the same in both groups. Mean serum LH and FSH levels were elevated for 34 h after GnRH agonist administration. In contrast, mean serum hCG levels were elevated for approximately 6 days after the administration of hCG, and serum FSH levels did not change. Mean luteal phase serum estradiol concentrations were lower in the GnRH agonist group than in the hCG group (P less than 0.02). No differences were observed in mean serum progesterone or PRL during the luteal phase or in the length of the luteal phase in the two groups. The mean number of oocytes retrieved and embryo number and quality did not differ between the two groups. Three of nine GnRH agonist cycles and none of nine hCG cycles resulted in clinical pregnancy (P = 0.1). The results of this study indicate that GnRH agonist is able to simulate a midcycle surge of gonadotropins, leading to follicular maturation and pregnancy. Further work is needed to determine whether there is any clinical advantage of GnRH agonist over hCG administration with regard to pregnancy rates.

Interleukin-1 stimulates human chorionic gonadotropin secretion by first trimester human trophoblast.

Interleukin-1 (IL-1) has been reported to stimulate LH, GH, ACTH, and TSH release from cultured pituitary cells. IL-1 also has been found to be secreted in significant amounts by placental macrophages. To determine the possible role of IL-1 within the placenta, we studied the effects of human recombinant IL-1 on hCG release by long term cultures of human first trimester trophoblast and the JAR (human choriocarcinoma) cell line. IL-1 in concentrations ranging from 10(-11)-10(-9) mol/L stimulated hCG release from trophoblast cultures. This stimulatory effect was not mediated by prostaglandin E2 (PGE2), as indicated by the inability of indomethacin to block this stimulation as well as a lack of IL-1 effect on PGE2 release by trophoblast cells. At these doses, IL-1 exerted no effect on hCG release by JAR cells. PGE2, when used in high concentrations (10(-6)-10(-5) mol/L), stimulated the release of hCG by the trophoblasts as well as by the JAR cells. Neither IL-1 nor PGE2 stimulated the proliferation, [3H]thymidine incorporation, or differentiation (syncytium formation) of trophoblast or JAR cells. These results suggest that IL-1 may be an important local regulator of hCG secretion by first trimester trophoblasts.

Evidence for increased dopaminergic and opioid activity in patients with hypothalamic hypogonadotropic amenorrhea.

PIP: The functional activity of endogenous opioids and dopamine (DA) was assessed by analyzing gonadotropin and prolactin responses to DA receptor antagonist (metoclopramide) and the opioid receptor antagonist (naloxone) in selected patients with hypothalamic hypogonadotropic amenorrhea and in normal cycling women. 8 amenorrheic and 9 normal women during the low estrogen (early follicular) phase of menstrual cycle were studied. Simultaneous blood sampling and agent infusion was performed via 2 indwelling catheters after an overnight fast. Those receiving metoclopramide (10 mg intravenous bolus) and those receiving naloxone (1.6 mg/h for 4 hours) had blood samples withdrawn at various intervals. The 8 amenorrheic patients weighed significantly less (P .01) and had significantly lower mean basal serum luteinizing hormone (LH) (P .0001) and prolactin (P .01) than normal women. Metoclopramide administered to normal women induced no significant changes in pituitary gonadotropin levels. In contrast, 4 of 8 hypogonadotropic amenorrhea patients responded to metoclopramide with a significant mean net change of LH (P .05); a concomitant rise in follicle stimulating hormone (FSH) was also seen but was not statistically significant. The other 4 showed no response, as in normal women. Prolactin response to metoclopramide was reduced uniformly in all 8 patients, independent of LH responders or nonresponders. Naloxone in normal women showed no response. However, a clear increment of mean LH in response to naloxone was observed in the same 4 hypogonadotropic amenorrhea patients who also responded to metoclopramide. Mean LH nearly doubled; FSH levels showed no significant changes. Those other 4 patients who had no response to metoclopramide also had no response to naloxone. The 4 LH nonresponders with significantly (P .01) lower mean basal prolactin levels compared with normal women showed greater than l00% increase in prolactin levels by Hour 3 of naloxone infusion; in contrast, the LH responders showed no changes in prolactin levels in response to naloxone infusion.^ieng