Alberto Revelli

Follicular fluid content and oocyte quality: from single biochemical markers to metabolomics

The assessment of oocyte quality in human in vitro fertilization (IVF) is getting increasing attention from embryologists. Oocyte selection and the identification of the best oocytes, in fact, would help to limit embryo overproduction and to improve the results of oocyte cryostorage programs. Follicular fluid (FF) is easily available during oocyte pick-up and theorically represents an optimal source on non-invasive biochemical predictors of oocyte quality. Unfortunately, however, the studies aiming to find a good molecular predictor of oocyte quality in FF were not able to identify substances that could be used as reliable markers of oocyte competence to fertilization, embryo development and pregnancy. In the last years, a well definite trend toward passing from the research of single molecular markers to more complex techniques that study all metabolites of FF has been observed. The metabolomic approach is a powerful tool to study biochemical predictors of oocyte quality in FF, but its application in this area is still at the beginning. This review provides an overview of the current knowledge about the biochemical predictors of oocyte quality in FF, describing both the results coming from studies on single biochemical markers and those deriving from the most recent studies of metabolomics

Cancer and fertility preservation: International recommendations from an expert meeting

In the last years, thanks to the improvement in the prognosis of cancer patients, a growing attention has been given to the fertility issues. International guidelines on fertility preservation in cancer patients recommend that physicians discuss, as early as possible, with all patients of reproductive age their risk of infertility from the disease and/or treatment and their interest in having children after cancer, and help with informed fertility preservation decisions. As recommended by the American Society of Clinical Oncology and the European Society for Medical Oncology, sperm cryopreservation and embryo/oocyte cryopreservation are standard strategies for fertility preservations in male and female patients, respectively; other strategies (e.g. pharmacological protection of the gonads and gonadal tissue cryopreservation) are considered experimental techniques. However, since then, new data have become available, and several issues in this field are still controversial and should be addressed by both patients and their treating physicians.In April 2015, physicians with expertise in the field of fertility preservation in cancer patients from several European countries were invited in Genova (Italy) to participate in a workshop on the topic of “cancer and fertility preservation”. A total of ten controversial issues were discussed at the conference. Experts were asked to present an up-to-date review of the literature published on these topics and the presentation of own unpublished data was encouraged. On the basis of the data presented, as well as the expertise of the invited speakers, a total of ten recommendations were discussed and prepared with the aim to help physicians in counseling their young patients interested in fertility preservation.Although there is a great interest in this field, due to the lack of large prospective cohort studies and randomized trials on these topics, the level of evidence is not higher than 3 for most of the recommendations highlighting the need of further research efforts in many areas of this field. The participation to the ongoing registries and prospective studies is crucial to acquire more robust information in order to provide evidence-based recommendations.

Effect of the method of conception and embryo transfer procedure on mid-gestation placenta and fetal development in an IVF mouse model

BACKGROUND Abnormal placentation is a potential mechanism to explain the increased incidence of low birthweight observed after IVF. This study evaluates, in a mouse model, whether the method of conception and embryo transfer affect placentation and fetal development. METHODS IVF blastocysts (CF1 x B6D2F1/J) were cultured in Whittens medium (IVF(WM), n = 55) or K modified simplex optimized medium with amino acids (IVF(KAA), n = 56). Embryos were transferred to the uteri of pseudo-pregnant recipients. Two control groups were created: unmanipulated embryos produced by natural mating (in vivo group, n = 64) and embryos produced by natural mating that were flushed from uterus and immediately transferred to pseudo-pregnant recipients (flushed blastocysts, FB group, n = 57). At gestation age 12.5 days, implantation sites were collected and fixed; fetuses and placentas were weighed and their developmental stage (DS) evaluated. Placental areas and vascular volume fractions were calculated; parametric statistics were applied as appropriate. RESULTS IVF fetuses showed a modest but significant delay in development compared with FB mice (P < 0.05). In addition, IVF conceptuses were consistently smaller than FB (P < 0.05). Importantly, these differences persisted when analyzing fetuses of similar DS. The placenta/fetus ratio was larger in the IVF group (IVF(WM) 0.95; IVF(KAA) = 0.90) than the FB group (0.72) (P < 0.05 for all comparisons). Gross morphology of the placenta and ratio labyrinth/fetal area were equivalent in the IVF and FB groups, as were percentage of fetal blood vessels, maternal blood spaces and trophoblastic components. CONCLUSIONS In vitro embryo culture affects fetal and placental development; this could explain the lower birthweight in IVF offspring.

Laparoscopic stripping of endometriomas negatively affects ovarian follicular reserve even if performed by experienced surgeons

In order to estimate the impact of laparoscopic stripping of endometriomas on the ovarian follicular reserve, 43 normo-ovulatory women were studied by endocrine (anti-Müllerian hormone (AMH), FSH, LH, inhibin B, oestradiol) and ultrasonographic (antral follicle count (AFC)) methods before surgery, and 3 and 9 months after surgery. The operation was performed by experienced laparoscopists, particularly aware of the need to avoid damaging the healthy part of the ovary. Serum AMH concentrations significantly decreased after the operation (1.4±0.2 ng/ml after 3 months and 1.3±0.3 ng/ml after 9 months versus 3.0±0.4 ng/ml before surgery; P<0.0001), whereas basal FSH, LH, oestradiol and inhibin B concentrations remained unchanged. The volume of the operated ovary significantly diminished after surgery (P<0.0001), whereas the AFC was not significantly altered. Overall, the data show that laparoscopic stripping of endometriomas reduces ovarian reserve. The significant decrease of AMH after surgery confirms that part of the healthy ovarian pericapsular tissue, containing primordial and preantral follicles, is removed or damaged despite all the surgical efforts to be atraumatic. This must be carefully considered when laparoscopic cystectomy surgery is scheduled for patients with no relevant symptoms besides infertility or with already small ovarian reserve.

In situ hybridization study of messenger RNA for estrogen receptor and immunohistochemical detection of estrogen and progesterone receptors in the human ovary

The expression of estrogen and progesterone receptors (ER and PR), as well as the presence of messenger RNA for estrogen receptor (ER-mRNA), were analyzed by immunohistochemistry and in situ hybridization, respectively, in the ovary of 25 healthy eumenorrheic women. Ovarian biopsies were taken in different phases of the menstrual cycle during laparotomy or operative laparoscopy performed for extraovarian benign diseases. A total of 126 follicles (105 primordial, 13 preantral and eight antral) and 50 corpora lutea (eight active and 42 atretic) was analyzed. Granulosa cells stained positively for ER, PR and ER-mRNA in 13.3, 9.5 and 17.1% of primordial follicles, respectively. The proportions of preantral and antral follicles with ER-positive granulosa cells were 23.1 and 37.5%, respectively; these follicles were positive for PR in 23.1 and 37.5% of cases, and for ER-mRNA in 30.7 and 37.5% of cases, respectively. For thecal cells, 38.5% of preantral and 37.5% of antral follicles were PR-positive, but no more than 25% stained positive for ER and ER-mRNA. Active corpora lutea stained positive for ER, PR and ER-mRNA in 50, 62.5 and 50% of cases, respectively. Corpora albicantes always stained negative. In all subjects the stroma surrounding both follicles and corpora lutea contained several fibroblast-like cells which stained positive for ER, PR and ER-mRNA. Oocytes and blood vessels stained negative in all cases. This study supports the hypothesis that estrogens and progesterone play a role in the intraovarian regulation of follicle growth from the first steps of follicle development, and participate in the regulation of corpus luteum.

Mutations and polymorphisms of the FSH receptor (FSHR) gene: clinical implications in female fecundity and molecular biology of FSHR protein and gene.

The portion of chromosome 2, including the gene codifying the receptor of FSH (FSHR gene), can display point mutations that cause variations in the amino acid sequence of the receptor protein (FSHR protein). Some of these structural changes affect the receptor functional properties that may be enhanced (activating mutations) or impaired (inactivating mutations). Activating mutations confer to FSHR a higher responsiveness to FSH, making it constitutively active even in the absence of the ligand, or render it able to nonspecifically respond to other tropic hormones (e.g., TSH). Inactivating mutations reduce the receptor’s function up to a total block, altering either the formation of the receptor-ligand complex, or FSH signal transduction. FSHR inactivating mutations may cause primary or secondary amenorrhea, infertility, and premature ovarian failure (POF), whereas activating mutations can predispose to ovarian hyperstimulation syndrome (OHSS) as a consequence of exogenous FSH administration, or with a spontaneous onset. Beside point mutations, FSHR gene polymorphisms at specific sites (e.g., codons 307 and 680) may influence FSHR protein responsiveness to exogenous FSH, and finally affect the effectiveness of in vitro fertilization (IVF) treatment as well as the likelihood of developing a severe OHSS as a consequence of superovulation. This review summarizes the current knowledge about the FSHR gene mutations and polymorphisms, illustrating in the first part their clinical consequences for female reproductive function. In the second part, it describes the techniques to study the FSHR gene sequence, and gives more details about the molecular biology of FSHR protein, of FSHR gene and its mutations. Target Audience: Obstetricians & Gynecologists, Family Physicians Learning Objectives: After reading this article, the reader should be able to list clinical disorders related to mutations in the follicle stimulating hormone receptor (FSHR) gene, explain the principles behind the study of FSHR mutations, and state possible future applications of knowledge of mutations in the FSHR gene in reproductive medicine.

Live birth after orthotopic grafting of autologous cryopreserved ovarian tissue and spontaneous conception in Italy

OBJECTIVE To describe a live birth obtained in Italy after autologous orthotopic transplantation of cryopreserved ovarian cortical tissue. DESIGN Case report. SETTING University department of gynecology and obstetrics, reproductive medicine and IVF unit. PATIENT(S) A 29-year-old patient affected by β-thalassemia (intermedia phenotype) who underwent chemotherapy and bone marrow transplantation at age 21 years, resulting in a complete precocious ovarian failure. INTERVENTION(S) Before being treated with chemotherapy (busulfan, cyclophosphamide, and cyclosporine) for bone marrow transplantation, the patient underwent laparoscopic sampling of ovarian cortical tissue that was frozen and cryopreserved in liquid nitrogen. Eight years later, the ovarian tissue was thawed and grafted during laparoscopy at an orthotopic site. MAIN OUTCOME MEASURE(S) Ultrasound and endocrine monitoring of the postgrafting restoration of ovarian function; conception, pregnancy, and live birth. RESULT(S) Three months after grafting, the decrease of circulating FSH levels and the parallel increase of E(2) levels demonstrated ovarian function restoration, which was confirmed by bidimensional ultrasound and color Doppler examinations. After some ovulatory cycles, the patient spontaneously conceived 16 months after transplantation. After 39 weeks of uneventful gestation, a healthy girl weighing 3,970 g was born. CONCLUSION(S) Autologous grafting of cryopreserved ovarian cortex at an orthotopic site may allow ovarian function restoration, spontaneous conception, and birth of a healthy baby.

Nitric oxide stimulates human sperm motility via activation of the cyclic GMP/protein kinase G signaling pathway

Nitric oxide (NO), a modulator of several physiological processes, is involved in different human sperm functions. We have investigated whether NO may stimulate the motility of human spermatozoa via activation of the soluble guanylate cyclase (sGC)/cGMP pathway. Sperm samples obtained by masturbation from 70 normozoospermic patients were processed by the swim-up technique. The kinetic parameters of the motile sperm-rich fractions were assessed by computer-assisted sperm analysis. After a 30-90  min incubation, the NO donor S-nitrosoglutathione (GSNO) exerted a significant enhancing effect on progressive motility (77, 78, and 78% vs 66, 65, and 62% of the control at the corresponding time), straight linear velocity (44, 49, and 48 μm/s vs 34, 35, and 35.5 μm/s), curvilinear velocity (81, 83, and 84 μm/s vs 68 μm/s), and average path velocity (52, 57, and 54 μm/s vs 40, 42, and 42 μm/s) at 5 μM but not at lower concentrations, and in parallel increased the synthesis of cGMP. A similar effect was obtained with the NO donor spermine NONOate after 30 and 60  min. The GSNO-induced effects on sperm motility were abolished by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (a specific sGC inhibitor) and mimicked by 8-bromo-cGMP (8-Br-cGMP; a cell-permeating cGMP analog); the treatment with Rp-8-Br-cGMPS (an inhibitor of cGMP-dependent protein kinases) prevented both the GSNO- and the 8-Br-cGMP-induced responses. On the contrary, we did not observe any effect of the cGMP/PRKG1 (PKG) pathway modulators on the onset of hyperactivated sperm motility. Our results suggest that NO stimulates human sperm motility via the activation of sGC, the subsequent synthesis of cGMP, and the activation of cGMP-dependent protein kinases.

A retrospective study on IVF outcome in euthyroid patients with anti-thyroid antibodies: effects of levothyroxine, acetyl-salicylic acid and prednisolone adjuvant treatments

BackgroundAnti-thyroid antibodies (ATA), even if not associated with thyroid dysfunction, are suspected to cause poorer outcome of in vitro fertilization (IVF).MethodsWe retrospectively analyzed: (a) the prevalence of ATA in euthyroid infertile women, (b) IVF outcome in euthyroid, ATA+ patients, and (c) the effect of adjuvant treatments (levothyroxine alone or associated with acetylsalicylic acid and prednisolone) on IVF results in ATA+ patients. One hundred twenty-nine euthyroid, ATA+ women undergoing IVF were compared with 200 matched, ATA-controls. During IVF cycle, 38 ATA+ patients did not take any adjuvant treatment, 55 received levothyroxin (LT), and 38 received LT +acetylsalicylic acid (ASA)+prednisolone (P).ResultsThe prevalence of ATA among euthyroid, infertile patients was 10.5%, similar to the one reported in euthyroid women between 18 and 45 years. ATA+ patients who did not receive any adjuvant treatment showed significantly poorer ovarian responsiveness to stimulation and IVF results than controls. ATA+ patients receiving LT responded better to ovarian stimulation, but had IVF results as poor as untreated ATA+ women. Patients receiving LT+ASA+P had significantly higher pregnancy and implantation rates than untreated ATA+ patients (PR/ET 25.6% and IR 17.7% vs. PR/ET 7.5% and IR 4.7%, respectively), and overall IVF results comparable to patients without ATA (PR/ET 32.8% and IR 19%).ConclusionThese observations suggest that euthyroid ATA+ patients undergoing IVF could have better outcome if given LT+ASA+P as adjuvant treatment. This hypothesis must be verified in further randomized, prospective studies.

IVF twins have similar obstetric and neonatal outcome as spontaneously conceived twins: a prospective follow-up study

Studies comparing the outcome of spontaneous versus IVF twin pregnancies report heterogeneous results. This may depend on differences in the studied populations and/or in the management approach to twin pregnancy. The aim of the present study was to compare both maternal and perinatal outcomes in dichorionic diamniotic twin pregnancies who where spontaneously conceived or originated by successful homologous IVF. In order to get homogeneous observations, monochorionic twin pregnancies and triplet pregnancies were excluded. Moreover, to avoid any possible bias deriving from differences in the obstetric management, all pregnancies were managed by the same team applying fixed obstetric protocols. The study included 223 twin pregnancies, 84 conceived by IVF and 139 spontaneously conceived. Overall, maternal and perinatal outcomes were similar in the two groups: no significant differences were observed as far as gestational age at delivery, birthweight, perinatal morbidity and mortality, and rate of malformations were concerned. The rate of Caesarean section was slightly, but not significantly, higher in IVF pregnancies. In conclusion, the outcome of IVF twin pregnancies is comparable to that of spontaneously conceived twin pregnancies, provided that the same management criteria are applied.