Manuel Fernández-Sánchez

The endometrial receptivity array for diagnosis and personalized embryo transfer as a treatment for patients with repeated implantation failure

OBJECTIVEnTo demonstrate the clinical value of the endometrial receptivity array (ERA) in patients with repeated implantation failure (RIF), for guiding their personalized embryo transfer (pET) as a novel therapeutic strategy.nnnDESIGNnProspective interventional multicenter clinical trial.nnnSETTINGnUniversity-affiliated infertility and private clinics.nnnPATIENT(S)nEighty-five RIF patients and 25 comparison patients.nnnINTERVENTION(S)nEndometrial sampling and pET guided by ERA.nnnMAIN OUTCOME MEASURE(S)nA receptive (R) or nonreceptive (NR) endometrial status according to ERA. Pregnancy (PR) and implantation (IR) rates after pET.nnnRESULT(S)nThe ERA test gave an R result of 74.1% in RIF patients versus 88% in control subjects. Clinical follow-up was possible in 29 RIF patients, in whom pET was performed, resulting in 51.7% PR and 33.9% IR. The IRs and PRs in the 6 months after the biopsy showed that pregnancy was not related to the local injury. Twenty-two RIF patients (25.9%) were NR, and in 15 of them a second ERA validated a displacement of the window of implantation (WOI). In eight of them, pET was performed on the day designated by the ERA, resulting in 50.0% PR and 38.5% IR. These results should be considered as preliminary.nnnCONCLUSION(S)nThere is an increased percentage of WOI displacement in RIF patients compared with comparison group patients, leading to the concept of pET as a therapeutic strategy. Rescue of NR patients by pET in a displaced WOI results in similar PR and IR.

Ovarian response to recombinant human follicle-stimulating hormone: a randomized, antimüllerian hormone–stratified, dose–response trial in women undergoing in vitro fertilization/intracytoplasmic sperm injection

OBJECTIVEnTo evaluate the dose-response relationship of a novel recombinant human FSH (rhFSH; FE 999049) with respect to ovarian response in patients undergoing IVF/intracytoplasmic sperm injection treatment; and prospectively study the influence of initial antimüllerian hormone (AMH) concentrations.nnnDESIGNnRandomized, controlled, assessor-blinded, AMH-stratified (low: 5.0-14.9 pmol/L [0.7-<2.1 ng/mL]; high: 15.0-44.9 pmol/L [2.1-6.3 ng/mL]) trial.nnnSETTINGnSeven infertility centers in four countries.nnnPATIENT(S)nTwo hundred sixty-five women aged ≤37 years.nnnINTERVENTION(S)nControlled ovarian stimulation with either 5.2, 6.9, 8.6, 10.3, or 12.1 μg of rhFSH, or 11 μg (150 IU) of follitropin alfa in a GnRH antagonist cycle.nnnMAIN OUTCOME MEASURE(S)nNumber of oocytes retrieved.nnnRESULT(S)nThe number of oocytes retrieved increased in an rhFSH dose-dependent manner, from 5.2 ± 3.3 oocytes with 5.2 μg/d to 12.2 ± 5.9 with 12.1 μg/d. The slopes of the rhFSH dose-response curves differed significantly between the two AMH strata, demonstrating that a 10% increase in dose resulted in 0.5 (95% confidence interval 0.2-0.7) and 1.0 (95% confidence interval 0.7-1.3) more oocytes in the low and high AMH stratum, respectively. Fertilization rate and blastocyst/oocyte ratio decreased significantly with increasing rhFSH doses in both AMH strata. No linear relationship was observed between rhFSH dose and number of blastocysts overall or by AMH strata. Five cases of ovarian hyperstimulation syndrome were reported for the three highest rhFSH doses and in the high AMH stratum.nnnCONCLUSION(S)nIncreasing rhFSH doses results in a linear increase in number of oocytes retrieved in an AMH-dependent manner. The availability of blastocysts is less influenced by the rhFSH dose and AMH level.nnnCLINICAL TRIAL REGISTRATION NUMBERnNCT01426386.

The non-ergot derived dopamine agonist quinagolide in prevention of early ovarian hyperstimulation syndrome in IVF patients: a randomized, double-blind, placebo-controlled trial

BACKGROUND Ovarian hyperstimulation syndrome (OHSS) seems to be induced by the ovarian release of vascular endothelial growth factor (VEGF), which increases vascular permeability. Dopamine agonists inhibit VEGF receptor phosphorylation and thereby decrease vascular permeability. METHODS A randomized, double-blind, placebo-controlled, multicentre study assessing three oral doses (50, 100, 200 µg/day) of the non-ergot derived dopamine agonist quinagolide started on the day of human chorionic gonadotrophin (hCG) and continued for 17–21 days without dose-titration in comparison to placebo in preventing moderate/severe early OHSS (onset ≤9 days after hCG administration) in 182 IVF patients with ≥20 but less than 30 follicles ≥10 mm. RESULTS The incidence of moderate/severe early OHSS was 23% (12/53) in the placebo group and 12% (6/51), 13% (7/52) and 4% (1/26) in the quinagolide 50, 100 and 200 µg/day groups, respectively. The moderate/severe early OHSS rate was significantly lower with all quinagolide groups combined compared with placebo [P = 0.019; OR = 0.28 (0.09–0.81)]. The incidence of ultrasound evidence of ascites among patients with no clinical pregnancy was significantly reduced from 31% (8/26) with placebo to 11% (8/70) with all quinagolide groups combined [P = 0.033; OR = 0.29 (0.10–0.88)], although there was no difference for those with clinical pregnancy. Quinagolide did not have a detrimental effect on pregnancy or live birth rates. The incidence of gastrointestinal and central nervous system adverse events increased with increasing doses of quinagolide. CONCLUSIONS Quinagolide appears to prevent moderate/severe early OHSS while not affecting treatment outcome. The effect is more marked in patients who did not achieve a clinical pregnancy. Quinagolide administered in high doses without dose-titration is associated with poor tolerability. ClinicalTrials.gov Identifier: NCT00329693.

Individualized versus conventional ovarian stimulation for in vitro fertilization: a multicenter, randomized, controlled, assessor-blinded, phase 3 noninferiority trial

OBJECTIVEnTo compare the efficacy and safety of follitropin delta, a new human recombinant FSH with individualized dosing based on serum antimüllerian hormone (AMH) and body weight, with conventional follitropin alfa dosing for ovarian stimulation in women undergoing IVF.nnnDESIGNnRandomized, multicenter, assessor-blinded, noninferiority trial (ESTHER-1).nnnSETTINGnReproductive medicine clinics.nnnPATIENT(S)nA total of 1,329 women (aged 18-40xa0years).nnnINTERVENTION(S)nFollitropin delta (AMH <15 pmol/L: 12xa0μg/d; AMH ≥15 pmol/L: 0.10-0.19xa0μg/kg/d; maximum 12xa0μg/d), or follitropin alfa (150 IU/d for 5xa0days, potential subsequent dose adjustments; maximum 450 IU/d).nnnMAIN OUTCOMES MEASURE(S)nOngoing pregnancy and ongoing implantation rates; noninferiority margins -8.0%.nnnRESULT(S)nOngoing pregnancy (30.7% vs. 31.6%; difference -0.9% [95% confidence interval (CI) -5.9% to 4.1%]), ongoing implantation (35.2% vs. 35.8%; -0.6% [95% CI -6.1% to 4.8%]), and live birth (29.8% vs. 30.7%; -0.9% [95% CI -5.8% to 4.0%]) rates were similar for individualized follitropin delta and conventional follitropin alfa. Individualized follitropin delta resulted in more women with target response (8-14 oocytes) (43.3% vs. 38.4%), fewer poor responses (fewer than four oocytes in patients with AMH <15 pmol/L) (11.8% vs. 17.9%), fewer excessive responses (≥15 or ≥20 oocytes in patients with AMH ≥15 pmol/L) (27.9% vs. 35.1% and 10.1% vs. 15.6%, respectively), and fewer measures taken to prevent ovarian hyperstimulation syndrome (2.3% vs. 4.5%), despite similar oocyte yield (10.0 ± 5.6 vs. 10.4 ± 6.5) and similar blastocyst numbers (3.3 ± 2.8 vs. 3.5 ± 3.2), and less gonadotropin use (90.0 ± 25.3 vs. 103.7 ± 33.6xa0μg).nnnCONCLUSION(S)nOptimizing ovarian response in IVF by individualized dosing according to pretreatment patient characteristics results in similar efficacy and improved safety compared with conventional ovarian stimulation.nnnCLINICAL TRIAL REGISTRATION NUMBERnNCT01956110.

Molecular and functional characterization of voltage-gated sodium channels in human sperm

BackgroundWe have investigated the expression of voltage-gated sodium channels in human spermatozoa and characterized their role in sperm motility.MethodsFreshly ejaculated semen was collected from thirty normozoospermic human donors, with each donor supplying 2 different samples. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence techniques were used to detect the mRNAs and proteins of interest. Sperm motility was measured by a computer-assisted sperm analysis system (CASA). Cytosolic free calcium was determined by fluorimetry in cells loaded with the fluorescent calcium indicator Fura-2.ResultsThe mRNAs that encode the different Nav alpha subunits (Nav1.1-1.9) were all expressed in capacitated human spermatozoa. The mRNAs of the auxiliary subunits beta1, beta3 and beta4 were also present. Immunofluorescence studies showed that, with the exception of Nav1.1 and Nav1.3, the Nav channel proteins were present in sperm cells and show specific and different sites of localization. Veratridine, a voltage-gated sodium channel activator, caused time- and concentration-dependent increases in progressive sperm motility. In sperm suspensions loaded with Fura-2, veratridine did not modify intracellular free calcium levels.ConclusionThis research shows the presence of voltage-gated sodium channels in human sperm and supports a role for these channels in the regulation of mature sperm function.

Expression of neurokinin B/NK3 receptor and kisspeptin/KISS1 receptor in human granulosa cells

STUDY QUESTIONnAre neurokinin B (NKB), NK3 receptor (NK3R), kisspeptin (KISS1) and kisspeptin receptor (KISS1R) expressed in human ovarian granulosa cells?nnnSUMMARY ANSWERnThe NKB/NK3R and kisspeptin/KISS1R systems are co-expressed and functionally active in ovarian granulosa cells.nnnWHAT IS KNOWN ALREADYnThe NKB/NK3R and KISS1/KISS1R systems are essential for reproduction. In addition to their well-recognized role in hypothalamic neurons, these peptide systems may contribute to the control of fertility by acting directly on the gonads, but such a direct gonadal role remains largely unknown.nnnSTUDY DESIGN, SIZE, DURATIONnThis study analyzed matched mural granulosa cells (MGCs) and cumulus cells (CCs) collected from preovulatory follicles of oocyte donors at the time of oocyte retrieval.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnThe samples were provided by 56 oocyte donor women undergoing ovarian stimulation treatment. Follicular fluid samples containing MGCs and cumulus-oocyte complexes were collected after transvaginal ultrasound-guided oocyte retrieval. RT-PCR, quantitative real-time PCR, immunocytochemistry and western blot were used to investigate the pattern of expression of the NKB/NK3R and KISS/KISS1R systems in MGCs and CCs. Intracellular free Ca(2+) levels, [Ca(2+)]i, in MGCs after exposure to NKB or KISS1, in the presence or not of tachykinin receptor antagonists, were also measured.nnnMAIN OUTCOME AND THE ROLE OF CHANCEnNKB/NK3R and KISS1/KISS1R systems were expressed, at the mRNA and protein levels, in MGCs and CCs, with significantly higher expression in CCs. Kisspeptin increased the [Ca(2+)]i in the cytosol of human MGCs while exposure to NKB failed to induce any change in [Ca(2+)]i. However, the [Ca(2+)]i response to kisspeptin was reduced in the presence of NKB. The inhibitory effect of NKB was only partially mimicked by the NK3R agonist, senktide and marginally suppressed by the NK3R-selective antagonist SB 222200. Yet, a cocktail of antagonists selective for the NK1, NK2 and NK3 receptors blocked the effect of NKB.nnnLIMITATIONS, REASONS FOR CAUTIONnThe granulosa and cumulus cells were obtained from oocyte donors undergoing ovarian stimulation, which in comparison with natural cycles, may have affected gene and protein expression in granulosa cells.nnnWIDER IMPLICATIONS OF THE FINDINGSnOur data demonstrate that, in addition to their indispensable effects at the central nervous system, the NKB/NK3R and kisspeptin/KISS1R systems are co-expressed and are functionally active in non-neuronal reproductive cells of the female gonads, the ovarian granulosa cells.nnnSTUDY FUNDING/ COMPETING INTERESTSnThis work was supported by grants from Ministerio de Economía y Competitividad (CTQ2011-25564 and BFI2011-25021) and Junta de Andalucía (P08-CVI-04185), Spain. J.G.-O., F.M.P., M.F.-S., N.P., A.C.-R., T.A.A., M.H., M.R., M.T.-S. and L.C. have nothing to declare.

Autocrine regulation of human sperm motility by tachykinins

BackgroundWe examined the presence and function of tachykinins and the tachykinin-degrading enzymes neprilysin (NEP) and neprilysin-2 (NEP2) in human spermatozoa.MethodsFreshly ejaculated semen was collected from forty-eight normozoospermic human donors. We analyzed the expression of substance P, neurokinin A, neurokinin B, hemokinin-1, NEP and NEP2 in sperm cells by reverse-transcriptase polymerase chain reaction (RT-PCR), western blot and immunocytochemistry assays and evaluated the effects of the neprilysin and neprilysin-2 inhibitor phosphoramidon on sperm motility in the absence and presence of tachykinin receptor-selective antagonists. Sperm motility was measured using WHO procedures or computer-assisted sperm analysis (CASA).ResultsThe mRNAs of the genes that encode substance P/neurokinin A (TAC1), neurokinin B (TAC3), hemokinin-1 (TAC4), neprilysin (MME) and neprilysin-2 (MMEL1) were expressed in human sperm. Immunocytochemistry studies revealed that tachykinin and neprilysin proteins were present in spermatozoa and show specific and differential distributions. Phosphoramidon increased sperm progressive motility and its effects were reduced in the presence of the tachykinin receptor antagonists SR140333 (NK1 receptor-selective) and SR48968 (NK2 receptor-selective) but unmodified in the presence of SR142801 (NK3 receptor-selective).ConclusionThese data show that tachykinins are present in human spermatozoa and participate in the regulation of sperm motility. Tachykinin activity is regulated, at least in part, by neprilysins.

The Voltage-Gated Sodium Channel Nav1.8 Is Expressed in Human Sperm

The role of Na+ fluxes through voltage-gated sodium channels in the regulation of sperm cell function remains poorly understood. Previously, we reported that several genes encoding voltage-gated Na+ channels were expressed in human testis and mature spermatozoa. In this study, we analyzed the presence and function of the TTX-resistant VGSC α subunit Nav1.8 in human capacitated sperm cells. Using an RT-PCR assay, we found that the mRNA of the gene SCN10A, that encode Na v1.8, was abundantly and specifically expressed in human testis and ejaculated spermatozoa. The Na v1.8 protein was detected in capacitated sperm cells using three different specific antibodies against this channel. Positive immunoreactivity was mainly located in the neck and the principal piece of the flagellum. The presence of Na v1.8 in sperm cells was confirmed by Western blot. Functional studies demonstrated that the increases in progressive motility produced by veratridine, a voltage-gated sodium channel activator, were reduced in sperm cells preincubated with TTX (10 μM), the Na v1.8 antagonist A-803467, or a specific Na v1.8 antibody. Veratridine elicited similar percentage increases in progressive motility in sperm cells maintained in Ca2+-containing or Ca2+-free solution and did not induce hyperactivation or the acrosome reaction. Veratridine caused a rise in sperm intracellular Na+, [Na+]i, and the sustained phase of the response was inhibited in the presence of A-803467. These results verify that the Na+ channel Na v1.8 is present in human sperm cells and demonstrate that this channel participates in the regulation of sperm function.

A comparison of live birth rates and cumulative ongoing pregnancy rates between Europe and North America after ovarian stimulation with corifollitropin alfa or recombinant follicle-stimulating hormone

OBJECTIVEnTo compare live birth rates after fresh embryo transfer (ET) and cumulative ongoing pregnancy rates after fresh ET and frozen-thawed (ET) between continents and overall after one treatment cycle with corifollitropin alfa or recombinant FSH.nnnDESIGNnDouble-blind, multicenter, randomized controlled trial.nnnSETTINGnFourteen centers in North America (NA); 20 in Europe (EU).nnnPATIENT(S)n804 NA patients and 702 EU patients.nnnINTERVENTION(S)nPatients >60 kg received a single dose of corifollitropin alfa or daily rFSH for the first 7 days of controlled ovarian stimulation.nnnMAIN OUTCOME MEASURE(S)nLive birth rates.nnnRESULT(S)nWithin each continent no differences were noted between the two treatment groups; however, between continents, the cumulative ongoing pregnancy rate and live birth rate were considerably higher in NA than in EU. The live birth rate in NA was 39.2% in both treatment groups compared with 31.5% and 28.8% in EU after corifollitropin alfa and rFSH treatment, respectively. Considering the number of embryos transferred, the live birth rate per ET was still higher in NA than in EU (42.7% v.s 36.8% with corifollitropin alfa and 41.6% vs. 30.9% with rFSH). Overall live birth rates after fresh ET were 35.6% and 34.4% (estimated difference 1.1% [95% confidence interval -3.7-5.8]), and the estimated cumulative live birth rates were 43.4% and 41.3% with corifollitropin alfa and rFSH, respectively.nnnCONCLUSION(S)nLive birth rates and cumulative pregnancy rates were higher in NA than in EU after treatment with either corifollitropin alfa or daily rFSH; both treatment protocols provided equal success rates. CLINICALTRIALS.GOV IDENTIFIERS: NCT00703014 and NCT00702273.

Monozygotic twinning after assisted reproductive technologies: a case report of asymmetric development and incidence during 19 years in an international group of in vitro fertilization clinics

OBJECTIVEnTo describe a case of monozygotic twinning with asymmetric development following a single fresh embryo transfer as part of an intracytoplasmic sperm injection (ICSI) treatment. Secondarily, to report the incidence of monozygotic twinning at the IVI (Instituto Valenciano de Infertilidad) clinics.nnnDESIGNnCase report.nnnSETTINGnPrivate fertility centers.nnnPATIENT(S)nA 33-year-old woman with a 2-year history of primary infertility.nnnINTERVENTION(S)nControlled ovarian hyperstimulation and ICSI treatment with single-embryo transfer.nnnMAIN OUTCOME MEASURE(S)nIncidence of monozygotic twinning at the IVI clinics.nnnRESULT(S)nWe report a twin pregnancy after a single-embryo transfer. Twins were dichorionic and diamniotic. One fetus had a 6-day delay in its growth compared with the other when observed by ultrasound. Two female infants were delivered, and despite presenting congenital diseases, they were successfully treated and evolved correctly. A subsequent DNA analysis confirmed that the infants were monozygotic. Furthermore, we estimated a monozygotic twinning rate of 1.17% at the IVI clinics, taking into account those cases in which two or more embryos with heart beats were observed by ultrasound scanning after single-embryo transfers.nnnCONCLUSION(S)nUltrasound scans performed during pregnancy suggested a possible dizygotic origin of the twins, but DNA analysis performed after birth established that they were monozygotic. Genetic analysis is the only valid tool to confirm if like-sex dichorionic twins are monozygotic or dizygotic.