Ricardo Pommer

The human corpus luteum: life cycle and function in natural cycles.

OBJECTIVE To summarize recent advances in the understanding of the endocrine signaling pathways between the hypothalamus, pituitary, and human corpus luteum (CL); to examine the major paracrine and autocrine mechanisms and the key genes and proteins involved in CL development, function, and regression in natural cycles; to review the endocrine and molecular response of the midluteal phase CL to in vivo administration of human chorionic gonadotropin (hCG); and to describe the ultrasonographic and Doppler evaluation of the ovary and endometrium throughout the luteal phase. DESIGN Published data in the literature, including the basic and clinical research studies of the authors. SETTING University-affiliated hospital and research centers. PATIENT(S) None. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Clinical and molecular analysis of human CL function. RESULT(S) The endocrine function of the subpopulations of luteal cells is critical for the maintenance of CL function, including neovacularization and steroid hormones production. We consider the key genes and proteins that favor development of luteal structure and function throughout the menstrual cycle and in our model of hCG treatment resembling early pregnancy. CONCLUSION(S) These data indicate that the functional lifespan of the CL depends on paracrine and autocrine mechanisms. Therefore, the significance of the key genes and proteins that we analyze in lutein cells during CL development, function, demise, and rescue by hCG is likely to bring new therapeutic applications for the management of fertility defects and the control of fertility.

Recent cigarette smoking and assisted reproductive technologies outcome

OBJECTIVE To assess the association between recent cigarette smoking (CS) in female and male partners and assisted reproduction technology (ART) outcomes. DESIGN Cohort prospective study. SETTING University ART program in Chile. PATIENT(S) One hundred sixty-six couples seeking pregnancy through ART. INTERVENTION(S) Follicular fluid (FF) and serum cotinine concentrations were measured in female partners. Self-reported CS data were collected through personal interviews. MAIN OUTCOME MEASURE(S) The association between female recent smoking, assessed by FF and serum cotinine concentrations, and ART outcomes, such as number of ova retrieved and implantation rates, and the association between self-reported male recent smoking and live birth rates. RESULT(S) A significant age-adjusted association between increased FF cotinine level and decreased number of ova retrieved was found. The male partners smoking habit significantly decreased the live birth rate from 21.1% to 7.8%. Serum cotinine concentrations paralleled those of FF. CONCLUSION(S) The hypothesis of a detrimental effect of recent female smoking over implantation rates is rejected. However, recent male smoking is associated with significantly decreased live birth rates even after adjusting for confounders. Female recent smoking was significantly associated with decreased number of retrieved ova.

P450-aromatase activity and expression in human testicular tissues with severe spermatogenic failure

There is evidence that impaired spermatogenesis is associated with an imbalance in the oestradiol/testosterone ratio and with Leydig cell (LC) dysfunction. In testis, P450-aromatase, encoded by CYP19, is responsible for the conversion of testosterone to oestradiol. The aims of this study were to quantify CYP19 mRNA expression, aromatase activity and protein localization, and to measure the oestradiol to testosterone ratio in testicular tissues of men with spermatogenic impairment. Twenty-four men with complete Sertoli cell-only syndrome (SCOS), 14 with focal SCOS, 14 with maturation arrest (MA), 8 with mixed atrophy and 30 controls with normal spermatogenesis were subjected to testicular biopsy. All subjects underwent a physical examination, cytogenetic and serum hormonal studies. Testicular CYP19 mRNA was quantified using real time RT-PCR. Testicular aromatase activity was measured using the (3)H(2)0 assay and protein expression was evaluated using immunohistochemistry. In cases, serum testosterone and oestradiol were normal, but the testosterone/LH ratio was lower compared with controls (p < 0.05). Aromatase was localized in the Leydig, Sertoli and germ cells of all tissues, although stronger intensity was observed in LC. Aromatase mRNA and activity were not altered in cases and correlated positively with LC number (r = 0.516 and r = 0.369; p < 0.008). The intratesticular oestradiol/testosterone ratio was elevated (p = 0.005) in complete SCOS patients compared with controls. In conclusion, testicular aromatase seems to be normal in most subjects with impaired spermatogenesis. However, an altered intratesticular oestradiol/testosterone ratio in some patients with complete SCOS suggests that aromatase is increased, which might contribute to Leydig cell dysfunction.

Cumulus cell apoptosis changes with exposure to spermatozoa and pathologies involved in infertility

OBJECTIVE To determine whether the incidence of apoptosis in mature oocyte cumulus cells changes after insemination related to infertility. DESIGN Prospective study. SETTING Public hospital and university. PATIENT(S) One hundred women undergoing in vitro fertilization and embryo transfer (IVF-ET). INTERVENTION(S) Collection of cumulus cells from IVF-ET cycles with different infertility etiologies. MAIN OUTCOME MEASURE(S) Detection of apoptosis in cumulus cells; fertilization, embryo quality, and pregnancy rate. RESULT(S) The incubation of cumulus-oocyte-complexes with spermatozoa led to an increase in cumulus cell apoptosis from 34.2 +/- 3.7 to 44.5 +/- 6.3%. After insemination, cumulus cells of poor quality embryos showed a statistically higher apoptotic rate versus cumulus cells of good quality embryos (61.5 +/- 6.4 vs. 40.6 +/- 3.9%). Cumulus cells arising from oocytes with >or=50% fertilization rates after insemination showed higher apoptosis rates did cumulus cells from oocytes with <50% fertilization rates (46.0 +/- 3.7 vs. 33.8 +/- 4.0%). Patients with endometriosis presented higher apoptotic rates before insemination (77.6 +/- 9.06%). Cumulus cells obtained after aspiration showed no differences in their apoptosis rates for the following factors: age of women, aspirated oocytes, estradiol level, fertilization rate, and embryo quality or pregnancy. The apoptotic profile from pregnant women was less than (but not statistically significantly different from) profiles from nonpregnant women. CONCLUSION(S) These results suggest that the incidence of apoptosis in cumulus cells is associated with exposure to spermatozoa and the cause of infertility.

Tumor necrosis factor-alpha activates nuclear factor-kappaB but does not regulate progesterone production in cultured human granulosa luteal cells.

Background. The role of tumor necrosis factor-α (TNF-α) in granulosa luteal cell function and steroidogenesis is still controversial. Our aim was to examine the steroidogenic response, together with the simultaneous expression and activation of nuclear factor-κB (NF-κB), in cultured human granulosa luteal cells (GLCs) following administration of TNF-α. Materials and methods. This prospective controlled study was conducted in the Human Reproduction Division at the Institute of Maternal and Child Research, Faculty of Medicine, University of Chile and the San Borja Arriarán Hospital, National Health Service, Santiago, Chile. GLCs were obtained from aspirates of follicles from women undergoing in vitro fertilization (IVF). Thirty-two women undergoing IVF for tubal-factor and/or male-factor infertility participated in this study. Protein levels of NF-κB, the NF-κB inhibitor IκBα and steroidogenic acute regulatory protein (StAR) were determined by Western blot and localization of NF-κB was studied by indirect immunofluorescence. Progesterone production was determined by radioimmunoassay. Results. TNF-α did not affect the expression of StAR protein or the synthesis of progesterone. NF-κB was expressed in the GLCs and activated by TNF-α, resulting in degradation of IκBα and mobilization of the p65 NF-κB subunit into the nucleus. Conclusions. These results indicate that TNF-α did not modulate steroidogenesis in cultured human GLCs. However, NF-κB was activated by TNF-α. Therefore the activation of NF-κB via the TNF-α pathway is likely associated with other preovulatory granulosa cell processes important for human ovarian function.

Neuroendocrine mechanism of anovulation in users of contraceptive subdermal implant of nomegestrol acetate (Uniplant).

OBJECTIVE To evaluate a nomegestrol acetate subdermal contraceptive implants (Uniplant; Thermex, Monaco) effect on the hypothalamus-pituitary-ovarian axis. DESIGN A prospective clinical trial. SETTING San Borja-Arriarán Clinical Hospital, University of Chile, School of Medicine. PATIENT(S) Normally cycling healthy women. INTERVENTION(S) Insertion of Uniplant. MAIN OUTCOME MEASURE(S) Luteinizing hormone pulse and endocrine profiles were assessed before, 48 hours after insertion, and after prolonged use of the implant. RESULT(S) Anovulation was noted in 100% of users in the first month. Seventy percent of subjects demonstrated follicular development with the absence of ovulation and an endocrine profile similar to the follicular phase: (LH pulse/8 hours 6.85 +/- 0.67, LH amplitude 3.54 +/- 0.65 mIU/mL (conversion factor to SI unit, 1.00), and E2 193 +/- 29.4 pg/mL (conversion factor to SI unit, 3.67), whereas 30% demonstrated no follicular activity with an endocrine profile similar to the luteal phase: (LH pulse/8 hours; 3.66 +/- 0.66, LH amplitude 5.76 +/- 1.73 mIU/mL, and E2 67.5 +/- 4 pg/mL. Clinical characteristics, serum gonadotropin concentration, and LH pulse characteristics failed to predict which subjects would initiate or remain devoid of follicular activity. CONCLUSION(S) Uniplant results in anovulation via two mechanisms: hypothalamic suppression in subjects who lack follicular development, and likely suppression of the pituitary LH surge in subjects who initiate follicular activity.

Decreased anti-Müllerian hormone concentration in follicular fluid of female smokers undergoing artificial reproductive techniques.

BACKGROUND Several reports indicate that women who smoke have an increased risk of failure to conceive compared with their non-smoker counterparts. Here, we assessed the effect of smoking during the Assisted Reproduction Therapy (ART) on a potential marker of ovarian reserve, anti-müllerian hormone (AMH) in the follicular fluid (FF). MATERIALS AND METHODS This was a cohort prospective study to assess the association between cigarette smoking and AMH concentrations in FF in fifty-six women undergoing their first ART cycle. Self-reported smoking status over time was also collected through personal interview. The main outcome measured was the association between current smoking and AMH concentrations in FF. Smoking status was assessed by FF cotinine concentrations. Analysis of covariance was performed to test statistical interaction between the main outcome and confounders. RESULTS The mean concentration of AMH in follicular fluid was significantly decreased among smokers (1.02±0.14 vs. 1.74±0.15, P<0.05). No statistical interaction was found between this difference in AMH concentrations and confounders like age and BMI. Thus, our data support the idea that AMH is decreased in active smokers across the fertile age. CONCLUSIONS The hypothesis of decreased AMH concentration in follicular fluid in female smokers was confirmed. The mechanisms through which cigarette smoking induces this fall in AMH are unknown and additional research is needed to improve our comprehension of the negative impact of smoking on ART outcomes.

DAX-1 and DAX-1A expression in human testicular tissues with primary spermatogenic failure

DAX-1 [dosage-sensitive sex reversal-adrenal hypoplasia congenital (AHC) critical region on the X chromosome gene 1; NR0B1] is an orphan nuclear receptor that acts as a transcriptional repressor in adrenal/gonadal development, steroidogenesis and probably spermatogenesis. An alternatively spliced form called DAX-1A (NR0B1A) has been described in several tissues including the testis, and in vitro studies have shown an inhibitory effect on DAX-1 transcriptional function. We aimed to study the mRNA and protein expression of DAX-1 in testicular tissues of 65 men with primary spermatogenic failure [complete Sertoli cell only syndrome (SCOS), focal SCOS, maturation arrest and mixed atrophy] compared with 33 controls with normal spermatogenesis. As a novel finding, we observed intense immunostaining, not only in the nucleus of Sertoli cells, but also in pachytene spermatocytes and round spermatids. The quantitative mRNA expression of DAX-1 and DAX-1A was similar between cases and controls and was not associated with the levels of gonadotrophins and steroids. Moreover, DAX-I transcript expression level was ∼750-fold higher than DAX-1A, and there was a strong positive correlation between them (r = 0.52; P< 0.001). We conclude that, in addition to Sertoli cells, DAX-1/DAX-1A is expressed in germ cells from spermatogonia to round spermatids. Besides, the similar mRNA expression of DAX-I and DAX-IA in testicular tissues from cases and controls does not support the involvement of DAX-1 in the etiology of primary spermatogenic failure. Finally, the low level of expression of the alternative transcriptional variant DAX-1A would not support its putative inhibitory function in vivo.

La criopreservación embrionaria en estadios más avanzados de desarrollo disminuye la frecuencia de pacientes con embriones criopreservados y el número de embriones criopreservados

Background: Multiple pregnancies are the main complication associated to assisted reproduction, due to the transfer of more than one embryo. Embryo cryopre-servation allows the sequential transfer of all generated embryos, thus diminishing the risk of multiple pregnancies. However, it leads to accumulation of cryopreserved embryos. To reduce their accumulation in our unit, we started to preserve embryos as blastocysts, that have a rate of successful pregnancies of approximately 40%. Aim: To perform a sensitivity analysis of this change of policy on the accumulation of embryos. Material and Methods: Records of 571 cycles of in vitro fertilization since 2007 were reviewed, assuming a transference rate of two embryos in women aged less than 35 years and three embryos in older women. The number of embryos that would be preserved as zygotes, eight cell stage or blastocysts, was analyzed. Results: Multiple component logistic regression analysis showed a 20% reduction in the odds ratio of cryopreservation per year of age. There was a 95% reduction in the ratio, when comparing the preservation of third and first day embryos and a 99% reduction when comparing preservation of embryos in blastocyst stage and first day embryos. Conclusions: Cryopreservation at blastocyst stage effectively decreased the frequency of embryo cryopreservation.

Do women offered assisted reproduction technologies have a higher incidence of gynecologic cancer? A systematic review and meta-analysis

The last two decades have seen an increase in the number of women diagnosed with infertility. The consequent growth in the use of assisted reproductive technologies (ART) calls for the determination of its long-term effects, including the risk of cancer. Many studies have attempted to answer this question, albeit with contradictory results. This review aimed to assess whether assisted reproductive technologies are associated with an increased risk of gynecological cancer. A search for papers in the literature was carried out on MEDLINE, TRIP DATABASE and NICE, resulting in 11 studies enrolling 3,900,231 patients altogether. Of these, 118,320 were offered ART. The incidence of gynecological cancer in the group offered ART was 0.6%, while the incidence in the group not offered ART was 2.1%. Taking all the studies into consideration, women offered ART were not at greater risk of having gynecological cancer; instead, a protective association was found.