A83-01 and DMH1 effects in the zebrafish spermatogonial niche: Unraveling the roles of TGF-ß and BMP signaling in the Fsh-mediated spermatogonial fate.

Transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) signaling has fundamental roles in the regulation of the stem cell niche for both embryonic and adult stem cells. In zebrafish, male germ stem cell niche is regulated by follicle-stimulating hormone (Fsh) through different members of the TGF-ß superfamily. On the other hand, the specific roles of TGF-ß and BMP signaling pathways are unknown in the zebrafish male germ stem cell niche. Considering this lack of information, the present study aimed to investigate the pharmacological inhibition of TGF-ß (A83-01) and BMP (DMH1) signaling pathways in the presence of recombinant zebrafish Fsh using testicular explants. We also reanalyzed single cell-RNA sequencing (sc-RNA-seq) dataset from adult zebrafish testes to identify the testicular cellular sites of smad expression, and to understand the physiological significance of the changes in smad transcript levels after inhibition of TGF-ß or BMP pathways. Our results showed that A83-01 potentiated the pro-stimulatory effects of Fsh on spermatogonial differentiation leading to an increase in the proportion area occupied by differentiated spermatogonia with concomitant reduction of type A undifferentiated (Aund) spermatogonia. In agreement, expression analysis showed lower mRNA levels for the pluripotency gene pou5f3, and increased expression of dazl (marker of type B spermatogonia and spermatocyte) and igf3 (pro-stimulatory growth factor) following the co-treatment with TGF-ß inhibitor and Fsh. Contrariwise, the inhibition of BMP signaling nullified the pro-stimulatory effects of Fsh, resulting in a reduction of differentiated spermatogonia and increased proportion area occupied by type Aund spermatogonia. Supporting this evidence, BMP signaling inhibition increased the mRNA levels of pluripotency genes nanog and pou5f3, and decreased dazl levels when compared to control. The sc-RNA-seq data unveiled a distinctive pattern of smad expression among testicular cells, primarily observed in spermatogonia (smad 2, 3a, 3b, 8), spermatocytes (smad 2, 3a, 8), Sertoli cells (smad 1, 3a, 3b), and Leydig cells (smad 1, 2). This finding supports the notion that inhibition of TGF-ß and BMP signaling pathways may predominantly impact cellular components within the spermatogonial niche, namely spermatogonia, Sertoli, and Leydig cells. In conclusion, our study demonstrated that TGF-ß and BMP signaling pathways exert antagonistic roles in the zebrafish germ stem cell niche. The members of the TGF-ß subfamily are mainly involved in maintaining the undifferentiated state of spermatogonia, while the BMP subfamily promotes spermatogonial differentiation. Therefore, in the complex regulation of the germ stem cell niche by Fsh, members of the BMP subfamily (pro-differentiation) should be more predominant in the niche than those belonging to the TGF-ß (anti-differentiation). Overall, these findings are not only relevant for understanding the regulation of germ stem cell niche but may also be useful for expanding in vitro the number of undifferentiated spermatogonia more efficiently than using recombinant hormones or growth factors.

The Ala307Thr polymorphism of the follicle-stimulating hormone receptor (FSHR) gene is associated with the dose of recombinant FSH received during IVF/ICSI treatment.

OBJECTIVE: Follicle-stimulating hormone (FSH) is essential for folliculogenesis, acting through the follicle-stimulating hormone receptor (FSHR) that is present on the membrane of granulosa cells. Polymorphisms in the FSHR gene may lead to an altered pattern of receptor expression on the cell surface or to changes in affinity for FSH. The aim of this prospective study was to detect any association between the follicle-stimulating hormone receptor (FSHR) gene Ala307Thr polymorphism (rs6165) and ovarian reserve, ovarian response or clinical results in IVF/ICSI treatment. METHODS: This prospective cohort study included 450 women who underwent IVF/ICSI cycles. DNA was extracted from peripheral blood, and the Ala307Thr FSHR polymorphism (rs6165) was genotyped using the TaqMan SNP genotyping assay. Participants were divided into three groups according to their Ala307Thr FSHR genotype: Thr/Thr (n:141), Thr/Ala (n=213) and Ala/Ala (n=96). The results were tested for associations with age, anti-Mullerian hormone (AMH) levels, antral follicle count (AFC), total dose of r-FSH, follicle size, number of retrieved oocytes, and clinical outcome of IVF/ICSI cycles. The statistical analyses were performed using Fisher's exact test and the Kruskal‒Wallis test. RESULTS: An association between the genotype of the FSHR (Ala307Thr) polymorphism and the dose of r-FSH was observed. Patients with the Ala/Ala genotype received a higher r-FSH dose than patients with the Ala/Thr (p=0.0002) and Thr/Thr (p=0.02) genotypes. No other correlation was observed. CONCLUSION: The Ala/Ala genotype was associated with the use of higher doses of recombinant FSH (r-FSH), suggesting that homozygosis of this allelic variant (Ala) provides lower sensitivity to r-FSH.

Gdnf Acts as a Germ Cell-Derived Growth Factor and Regulates the Zebrafish Germ Stem Cell Niche in Autocrine- and Paracrine-Dependent Manners.

Glial cell line-derived neurotrophic factor (GDNF) and its receptor (GDNF Family Receptor α1-GFRα1) are well known to mediate spermatogonial stem cell (SSC) proliferation and survival in mammalian testes. In nonmammalian species, Gdnf and Gfrα1 orthologs have been found but their functions remain poorly investigated in the testes. Considering this background, this study aimed to understand the roles of the Gdnf-Gfrα1 signaling pathway in zebrafish testes by combining in vivo, in silico and ex vivo approaches. Our analysis showed that zebrafish exhibit two paralogs for Gndf (gdnfa and gdnfb) and its receptor, Gfrα1 (gfrα1a and gfrα1b), in accordance with a teleost-specific third round of whole genome duplication. Expression analysis further revealed that both ligands and receptors were expressed in zebrafish adult testes. Subsequently, we demonstrated that gdnfa is expressed in the germ cells, while Gfrα1a/Gfrα1b was detected in early spermatogonia (mainly in types Aund and Adiff) and Sertoli cells. Functional ex vivo analysis showed that Gdnf promoted the creation of new available niches by stimulating the proliferation of both type Aund spermatogonia and their surrounding Sertoli cells but without changing pou5f3 mRNA levels. Strikingly, Gdnf also inhibited late spermatogonial differentiation, as shown by the decrease in type B spermatogonia and down-regulation of dazl in a co-treatment with Fsh. Altogether, our data revealed that a germ cell-derived factor is involved in maintaining germ cell stemness through the creation of new available niches, supporting the development of spermatogonial cysts and inhibiting late spermatogonial differentiation in autocrine- and paracrine-dependent manners.

Impact of Intracytoplasmic Morphologically Selected Sperm Injection (IMSI) on Birth Defects: A Systematic Review and Meta-Analysis.

OBJECTIVE: It is generally accepted that the incidence of birth defects in spontaneously conceived children ranges between 2.0-4.0%. However, several studies have shown that babies born after assisted reproductive technology (ART) procedures tend to present more congenital malformations than naturally conceived children, with 6.5% of the children born after intracytoplasmic sperm injection (ICSI) presenting birth defects. The use of high magnification sperm selection before ICSI was introduced in the early 2000s to allow the identification of spermatozoa with low risk of sperm DNA damage. Intracytoplasmic morphologically selected sperm injection (IMSI) is expected to change the incidence of congenital malformations, although data on the incidence of birth defects in children conceived after IMSI are still scarce. METHODS: A systematic review based on searches performed in electronic databases (PubMed, EMBASE, Web of Science, SCOPUS, and Cochrane Central Register of Controlled Trials) including articles published by February 2021 was conducted to identify trials comparing the neonatal outcomes of ICSI and IMSI. The outcome measured was the rate of birth defects in children born after ICSI or IMSI. Three trials were included as targets for data extraction and meta-analysis. RESULTS: Our meta-analysis included 3907 children conceived after IMSI (1280) or ICSI (2627). The incidence of birth defects was statistically different, with 2.5% (32/1280) in IMSI and 4.5% (119/2627) in ICSI (RR=0.59; 95% CI=0.40-0.87; p=0.007). The results demonstrated that IMSI decreased the incidence of structural defects compared to ICSI - 2.2% (18/830) vs. 3.8% (78/2049) - in a statistically significant manner (RR=0.58; 95%CI=0.35-0.96; p=0.04). No significant difference was observed in chromosomal abnormalities (Trisomy 13; 18; 21 and Triple X) between children conceived after IMSI (8/830) or ICSI (19/2049) (RR=1.07; 95%CI=0.47-2.43; p=0.87). CONCLUSIONS: IMSI seems to be an effective tool at reducing the incidence of structural defects compared to ICSI. However, IMSI does not change the incidence of chromosomal abnormalities.

Molecular characterization and expression analysis of anti-Müllerian hormone in common carp (Cyprinus carpio) adult testes.

Anti-Müllerian hormone (Amh) is a member of the transforming growth factor-ß (Tgf-ß) superfamily required in the regression of Müllerian ducts during gonadal sex differentiation of higher vertebrates. Teleost fish lack Müllerian ducts, but identified Amh orthologs have been shown to exert crucial functions during sex determination and differentiation of several species of teleosts. However, the function of Amh during gametogenesis in adult fish remains poorly investigated. Therefore, to expand present knowledge on the role of Amh in teleosts, the present study aimed to isolate and clone full-length amh cDNA in the common carp, Cyprinus carpio, and examine its expression levels throughout the male reproductive cycle and in response to different hormone treatments of testicular explants. Molecular cloning and characterization showed that the common carp Amh precursor amino acid sequence shared common features to other fish Amh precursors, including a conserved C-terminus (Tgf-ß domain) and a double proteolytic cleavage site (R-X-X-R-X-X-R) upstream to the Tgf-ß domain. Expression analysis showed amh dimorphic expression in the adult gonads with higher expression in the testes than ovaries. In testes, amh mRNA was detected in Sertoli cells contacting different types of germ cells, although the expression was greatest in Sertoli cells associated with type A undifferentiated spermatogonia. Expression analysis during the reproductive cycle showed that amh transcripts were down-regulated during the developing phase, which is characterized by an increased proliferation of type A undifferentiated spermatogonia and Sertoli cells and appearance of spermatocytes (meiosis) in the testes. Furthermore, ex vivo experiments showed that a 7 day exposure to Fsh or estrogens was required to decrease amh mRNA levels in common carp testicular explants. In summary, this study provided information on the molecular characterization and transcript abundance of amh in common carp adult testes. Altogether, these data will be useful for further investigations on sex determination and differentiation in this species, and also to improved strategies for improved carp aquaculture, such as inhibiting precocious maturation of males.

Noninvasive Preimplantation Genetic Testing for Aneuploidy (niPGT-A): The first Brazilian baby.

Recently, a new technology known as the Noninvasive Preimplantation Genetic Testing for Aneuploidy (niPGT-A) emerged, using cell-free DNA present in the spent culture media of human blastocysts. Unlike PGT-A, in which only trophectoderm cells are used, niPGT-A reflects the ploidy state of these cells and internal cell mass, suggesting that this new technology may be less prone to error, being more reliable than the invasive test. The aim of the present study was to report the first occurrence of childbirth following niPGT-A in Brazil.

Association between estrogen receptor 1 (ESR1) and leukemia inhibitory factor (LIF) polymorphisms can help in the prediction of recurrent implantation failure.

OBJECTIVE: To investigate an association between polymorphisms related to the implantation process that together could help in the prediction of recurrent implantation failure (RIF). DESIGN: Cohort study. SETTING: Private fertility center and reproductive genetics laboratory. PATIENT(S): Forty-four women presenting RIF, who were included in study group (RIF group), and two control groups, one with 63 women who were attended at our service and became pregnant after the first IVF/intracytoplasmic sperm injection attempt (control group I) and other with 65 fertile women who had at least two children without any treatment and no history of miscarriage (control group II). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Genotyping was performed in the intron region of TP63, VEGFA, MMP2, ESR1, and ESR2 genes and in the 3' untranslated region of the LIF gene on genomic DNA using real-time polymerase chain reaction. RESULT(S): The presence of ESR1/AA (rs12199722) and LIF/GT (rs929271) genotypes was more frequent in the RIF group, leading to a 7.9-fold increase in the chance of women presenting with RIF when compared with women who became pregnant on their first cycle of IVF/intracytoplasmic sperm injection and a 2.8-fold increase when compared with women who became pregnant without treatment. CONCLUSION(S): The association between ESR1 and LIF polymorphisms can help in the prediction of RIF.

Effects of GnRH and the dual regulatory actions of GnIH in the pituitary explants and brain slices of Astyanax altiparanae males.

The pituitary gonadotropins, Fsh (follicle-stimulating hormone) and Lh (luteinizing hormone), regulate testicular development and functions in all vertebrates. At the pituitary, different signaling systems regulate the synthesis and secretion of the gonadotropins, such as the hypothalamic neuropeptides GnRH (gonadotropin-releasing hormone) and GnIH (gonadotropin-inhibitory hormone). While GnRH exerts stimulatory roles, the actions of GnIH remain controversial for many teleost species. Therefore, the aim of this study was to evaluate the in vitro effects of chicken GnRH2 (cGnRH2) and zebrafish GnIH-3 (zGnIH-3) on the male gonadotropin and GnRH system expression using pituitary explants and brain slices from a neotropical species with economical and ecological relevance, Astyanax altiparanae. Our results showed that in males, cGnRH2 increased fshb and lhb mRNA levels in the pituitary explants. Interestingly, zGnIH-3 has no effect on basal gonadotropin expression, however zGnIH-3 decreased the cGnRH2-induced fshb and lhb transcripts in male pituitary explants. In the male brain slices, zGnIH-3 showed stimulatory effects, increasing gnrh2 mRNA levels. Overall, our results suggested that GnIH seems to have dual regulatory actions on gonadotropin and GnRH2 expression of A. altiparanae males. This study provided basic information on endocrine regulation of A. altiparanae reproduction, and the obtained results will expand our knowledge, improving the reproductive management of this economically important freshwater species.

Randomized comparison of two commercial culture media (Cook and Vitrolife) for embryo culture after IMSI.

OBJECTIVE: A variety of studies randomizing women/cycles or oocytes/embryos has been carried out to compare different culture media for culturing embryos up to cleavage or blastocyst stages showing controversial results. A recent systematic review suggested that data in the literature are insufficient to conclude the best culture medium for embryo quality, pregnancy and implantation. The objective of this study was to evaluate whether there is any difference between two commercial culture media regarding clinical outcomes after IMSI cycles. METHODS: A total of 120 patients, ≤39 years of age, undergoing ART treatment submitted to the IMSI program were prospectively broken down and randomized into two groups: Group I (Cook media) and Group II (Vitrolife media). RESULTS: Our data demonstrated that there was no difference using all the media from Cook or all the media from Vitrolife, for culturing embryos till day 2, in the bench incubator at low O2 concentration, in relation to fertilization, embryo quality, pregnancy and implantation rates (p>0.05). CONCLUSION: Both culture media used, Cook medium and Vitrolife medium, for the IMSI procedure and for later embryo culture with transfer on the second day, are equally effective and can be used depending on the ease and availability of acquisition.

The effects of male age on sperm DNA damage: an evaluation of 2,178 semen samples.

OBJECTIVE: This study aimed to evaluate the effects of male age on sperm DNA damage. METHODS: This cross-sectional study included semen samples collected from 2,178 men seen at an infertility clinic. For DNA integrity analysis, the proportions of spermatozoa showing DNA fragmentation (TUNEL assay), abnormal chromatin packaging/underprotamination (chromomycin A3), abnormal mitochondrial membrane potential (MMP/MitoTracker Green), and apoptosis (annexin V) were recorded. For group comparisons, enrolled subjects were divided into three groups based on their ages: ≤35 years; 36-44 years; and ≥45 years. The associations between age and sperm parameters were assessed using Spearman's rank correlation coefficient. RESULTS: Although aging did not affect sperm apoptosis (p>.05), sperm DNA fragmentation and MMP deteriorated significantly with age (p<.05). Chromatin packaging/protamination improved significantly with age (p<.05). CONCLUSION: Sperm DNA fragmentation worsened with age and was apparently associated with mitochondrial damage. The age-related increase in sperm DNA damage suggests that delaying childbearing, not only in women but also in men, might jeopardize a couple's reproductive capacity. The increase seen in chromatin packaging might represent a protective feature for DNA. However, additional studies must be performed to confirm the results concerning chromatin packaging/protamination.

Characterization of vasa homolog in a neotropical catfish, Jundiá (Rhamdia quelen): Molecular cloning and expression analysis during embryonic and larval development.

We have characterized the full-length vasa cDNA from Jundiá, Rhamdia quelen (Heptapteridae, Siluriformes). vasa encodes a member of the DEAD-box protein family of ATP-dependent RNA helicases. This protein is highly conserved among different organisms and its role is associated with RNA metabolism. In the majority of the investigated species, vasa is restricted to the germ cell lineage and its expression has been used to study germline development in many organisms, including fish. The deduced R. quelen vasa amino acid sequence displayed high similarity with Vasa protein sequences from other organisms, and did not cluster with PL10 or P68 DEAD-box protein subfamilies. We also reported that there is no other isoform for vasa mRNA in R. quelen gonads. Expression analysis by RT-PCR and qPCR showed vasa transcripts exclusively expressed in the germ cells of R. quelen gonads. R. quelen vasa mRNA was maternally inherited, and was detected in the migrating primordial germ cells (PGCs) until 264 h post-fertilization during embryonic and larval development. This work has characterized for the first time the full-length R. quelen vasa cDNA, and describes its expression patterns during R. quelen embryonic and larval development. Our results will contribute to the basic reproductive biology of this native species, and will support studies using vasa as a germ cell marker in different biotechnological studies, such as germ cell transplantation.

Single versus sequential culture medium: which is better at improving ongoing pregnancy rates? A systematic review and meta-analysis.

This study aimed to evaluate if single medium is better than sequential medium at improving ongoing pregnancy rates in patients undergoing assisted reproductive technology (ART) procedures. The data featured in this meta-analysis were extracted from four randomized controlled trials yielded from a systematic search carried out on electronic databases. The primary endpoint was ongoing pregnancy rate. Secondary endpoints included clinical pregnancy and miscarriage rates. The endpoints for ongoing pregnancy rate were also analyzed based on the time at which the embryo transfers were performed: cleavage stage (day 2/3) and/or blastocyst stage (day 5/6). There were no significant differences between single and sequential medium for clinical pregnancy (RR=1.09; 95%CI=0.83-1.44; p=0.53), ongoing pregnancy (RR=1.11; 95%CI=0.87-1.40; p=0.39), or miscarriage rates (RR=0.89; 95%CI=0.44-1.81; p=0.74). No significant difference was found for ongoing pregnancy rate (RR=1.29; 95%CI=0.93-1.78; p=0.12) between single and sequential medium when only trials in which embryos were transferred at the blastocyst stage were included. In conclusion, the choice of embryo culture approach - single or sequential medium - did not affect the ongoing pregnancy rates of patients undergoing ART cycles.

Key performance indicators score (KPIs-score) based on clinical and laboratorial parameters can establish benchmarks for internal quality control in an ART program.

OBJECTIVE: KPIs have been employed for internal quality control (IQC) in ART. However, clinical KPIs (C-KPIs) such as age, AMH and number of oocytes collected are never added to laboratory KPIs (L-KPIs), such as fertilization rate and morphological quality of the embryos for analysis, even though the final endpoint is the evaluation of clinical pregnancy rates. This paper analyzed if a KPIs-score strategy with clinical and laboratorial parameters could be used to establish benchmarks for IQC in ART cycles. METHODS: In this prospective cohort study, 280 patients (36.4±4.3years) underwent ART. The total KPIs-score was obtained by the analysis of age, AMH (AMH Gen II ELISA/pre-mixing modified, Beckman Coulter Inc.), number of metaphase-II oocytes, fertilization rates and morphological quality of the embryonic lot. RESULTS: The total KPIs-score (C-KPIs+L-KPIs) was correlated with the presence or absence of clinical pregnancy. The relationship between the C-KPIs and L-KPIs scores was analyzed to establish quality standards, to increase the performance of clinical and laboratorial processes in ART. The logistic regression model (LRM), with respect to pregnancy and total KPIs-score (280 patients/102 clinical pregnancies), yielded an odds ratio of 1.24 (95%CI = 1.16-1.32). There was also a significant difference (p<0.0001) with respect to the total KPIs-score mean value between the group of patients with clinical pregnancies (total KPIs-score=20.4±3.7) and the group without clinical pregnancies (total KPIs-score=15.9±5). Clinical pregnancy probabilities (CPP) can be obtained using the LRM (prediction key) with the total KPIs-score as a predictor variable. The mean C-KPIs and L-KPIs scores obtained in the pregnancy group were 11.9±2.9 and 8.5±1.7, respectively. Routinely, in all cases where the C-KPIs score was ≥9, after the procedure, the L-KPIs score obtained was ≤6, a revision of the laboratory procedure was performed to assess quality standards. CONCLUSION: This total KPIs-score could set up benchmarks for clinical pregnancy. Moreover, IQC can use C-KPIs and L-KPIs scores to detect problems in the clinical-laboratorial interface.

Characterization of Gnrh/Gnih elements in the olfacto-retinal system and ovary during zebrafish ovarian maturation.

Gonadotropin releasing hormone (GnRH) is one of the key players of brain-pituitary-gonad axis, exerting overall control over vertebrate reproduction. In zebrafish, two variants were characterized and named as Gnrh2 and Gnrh3. In this species, Gnrh3, the hypohysiotropic form, is expressed by neurons of the olfactory-retinal system, where it is related with food detection, intra/interspecific recognition, visual acuity and retinal processing modulation. Previous studies have reported the presence of Gnrh receptors in the zebrafish retina, but not yet in the zebrafish olfactory epithelium. The current study analyzed the presence of gnrh2 and gnrh3, their receptors (gnrhr 1,2,3 and 4) and gnih (gonadotropin inhibitory hormone) transcripts, as well as the Gnrh3 protein in the olfactory epithelium (OE), olfactory bulb (OB), retina and ovary during zebrafish ovarian maturation. We found an increase of gnrh receptors transcripts in the OE at the final stages of ovarian maturation. In the OE, Gnrh3 protein was detected in the olfactory receptor neurons cilia and in the olfactory nerve fibers. Interestingly, in the OB, we found an inverse expression pattern between gnih and gnrh3. In the retina, gnrhr4 mRNA was found in the nuclei of amacrine, bipolar, and ganglion cells next to Gnrh3 positive fibers. In the ovary, gnrh3, gnrhr2 and gnrhr4 transcripts were found in perinucleolar oocytes, while gnih in oocytes at the cortical alveolus stage. Our results suggested that Gnrh/Gnih elements are involved in the neuromodulation of the sensorial system particularly at the final stages of maturation, playing also a paracrine role in the ovary.

Association between leukaemia inhibitory factor gene polymorphism and pregnancy outcomes after assisted reproduction techniques.

Certain gene polymorphisms are associated with implantation failure and pregnancy loss. Studies of leukaemia inhibitory factor (LIF) gene polymorphisms are scarce. The LIF single nucleotide polymorphism (SNP) thymine (T)/guanine (G) (rs929271) was studied in women to determine whether an association existed with pregnancy outcomes after intracytoplasmic sperm injection (ICSI); 411 women who underwent ICSI were recruited. DNA was extracted from the peripheral blood, and the LIF gene SNP T/G (rs929271) was genotyped using real-time polymerase chain reaction. Participants were divided into three groups according to their LIF genotype: T/T (n = 168), T/G (n = 202) and G/G (n = 41). All IVF and ICSI procedures were carried out under the same clinical and laboratory conditions. The ICSI cumulative results (from fresh plus frozen cycles) of each genotype group were analysed. The G/G genotype in women was associated with a higher implantation rate (T/T: 15.9%, T/G: 16.2%, G/G: 27.0%; P < 0.05), ongoing pregnancy rate/patient (T/T: 31.5%, T/G: 36.1%, G/G: 53.7%; P < 0.05) and ongoing pregnancy rate/transfer (T/T: 18.5%, T/G: 20.2%, G/G: 36.7%; P < 0.05). LIF SNP T/G (rs929271) seems to be a susceptibility biomarker capable of predicting implantation efficiency and pregnancy outcomes.

A new ovarian response prediction index (ORPI): implications for individualised controlled ovarian stimulation.

BACKGROUND: The objective was to present a new ovarian response prediction index (ORPI), which was based on anti-Müllerian hormone (AMH) levels, antral follicle count (AFC) and age, and to verify whether it could be a reliable predictor of the ovarian stimulation response. METHODS: A total of 101 patients enrolled in the ICSI programme were included. The ORPI values were calculated by multiplying the AMH level (ng/ml) by the number of antral follicles (2-9 mm), and the result was divided by the age (years) of the patient (ORPI=(AMH x AFC)/Patient age). RESULTS: The regression analysis demonstrated significant (P<0.0001) positive correlations between the ORPI and the total number of oocytes and of MII oocytes collected. The logistic regression revealed that the ORPI values were significantly associated with the likelihood of pregnancy (odds ratio (OR): 1.86; P=0.006) and collecting greater than or equal to 4 oocytes (OR: 49.25; P<0.0001), greater than or equal to 4 MII oocytes (OR: 6.26; P<0.0001) and greater than or equal to 15 oocytes (OR: 6.10; P<0.0001). Regarding the probability of collecting greater than or equal to 4 oocytes according to the ORPI value, the ROC curve showed an area under the curve (AUC) of 0.91 and an efficacy of 88% at a cut-off of 0.2. In relation to the probability of collecting greater than or equal to 4 MII oocytes according to the ORPI value, the ROC curve had an AUC of 0.84 and an efficacy of 81% at a cut-off of 0.3. The ROC curve for the probability of collecting greater than or equal to 15 oocytes resulted in an AUC of 0.89 and an efficacy of 82% at a cut-off of 0.9. Finally, regarding the probability of pregnancy occurrence according to the ORPI value, the ROC curve showed an AUC of 0.74 and an efficacy of 62% at a cut-off of 0.3. CONCLUSIONS: The ORPI exhibited an excellent ability to predict a low ovarian response and a good ability to predict a collection of greater than or equal to 4 MII oocytes, an excessive ovarian response and the occurrence of pregnancy in infertile women. The ORPI might be used to improve the cost-benefit ratio of ovarian stimulation regimens by guiding the selection of medications and by modulating the doses and regimens according to the actual needs of the patients.

Motile sperm organelle morphology examination (MSOME): intervariation study of normal sperm and sperm with large nuclear vacuoles.

BACKGROUND: Although the motile sperm organelle morphology examination (MSOME) was developed only as a selection criterion, its application as a method for classifying sperm morphology may represent an improvement in evaluation of semen quality, with potential clinical repercussions. The present study aimed to evaluate individual variations in the motile sperm organelle morphology examination (MSOME) analysis after a time interval. METHODS: Two semen samples were obtained from 240 men from an unselected group of couples undergoing infertility investigation and treatment. Mean time interval between the two semen evaluations was 119+/-102 days. No clinical or surgical treatment was realized between the two observations. Spermatozoa were analyzed at greater than or equal to 8400x magnification by inverted microscope equipped with DIC/Nomarski differential interference contrast optics. At least 200 motile spermatozoa per semen sample were evaluated and percentages of normal spermatozoa and spermatozoa with large nuclear vacuoles (LNV/one or more vacuoles occupying>50% of the sperm nuclear area) were determined. A spermatozoon was classified as morphologically normal when it exhibited a normal nucleus (smooth, symmetric and oval nucleus, width 3.28+/-0.20 microm, length 4.75+/-0.20 microm/absence of vacuoles occupying>4% of nuclear area) as well as acrosome, post-acrosomal lamina, neck and tail, besides not presenting cytoplasm around the head. One examiner, blinded to subject identity, performed the entire study. RESULTS: Mean percentages of morphologically normal and LNV spermatozoa were identical in the two MSOME analyses (1.6+/-2.2% vs. 1.6+/-2.1% P=0.83 and 25.2+/-19.2% vs. 26.1+/-19.0% P=0.31, respectively). Regression analysis between the two samples revealed significant positive correlation for morphologically normal and for LNV spermatozoa (r=0.57 95% CI:0.47-0.65 P<0.0001 and r=0.50 95% CI:0.38-0.58 P<0.0001, respectively). CONCLUSIONS: The significant positive correlation and absence of differences between two sperm samples evaluated after a time interval with respect to normal morphology and LNV spermatozoa indicated that MSOME seems reliable (at least for these two specific sperm forms) for analyzing semen. The present result supports the future use of MSOME as a routine method for semen analysis.