The relationship between primary ovarian insufficiency and gene variations: a prospective case-control study.

Around 70 percent of cases of Primary Ovarian Insufficiency (POI) etiology remain unexplained. The aim of our study is to contribute to the etiology and genetic background of POI. A total of 37 POI patients and 30 women in the reproductive period were included in this prospective, case-control study between August 2020 and December 2021. The women were examined for 36 genes with next-generation sequencing (NGS) panel. Gene variations were detected in 59.5 percent of the patients in the case group. FSHR p.S680N (rs6166, c.2039 G>A) and FSHR p.A307T (rs6165, c.919 G>A) gene variants, which are most frequently located in exon 10 of the FSHR gene, were detected in both groups. Although it was not found that these gene variants were significantly different between the groups, it was also found that they were significantly different in POI patients under 30 years of age and in those with a family history of POI. Variations were detected in 12 genes in POI patients. Two gene variants (FGFR1 [c.386A>C, rs765615419] and KISS1 [c.58 G>A, rs12998]) were detected in both groups, and the remaining gene variants were detected only in POI patients. No differences were detected between the groups in terms of gene variations. However, the gene variations detected only in POI patients may play a role in the etiology of POI.

Paeoniflorin Alleviates Cisplatin-Induced Diminished Ovarian Reserve by Restoring the Function of Ovarian Granulosa Cells via Activating FSHR/cAMP/PKA/CREB Signaling Pathway.

Paeoniflorin (PAE) is the main active compound of Radix Paeoniae Rubra (a valuable traditional Chinese medicine and a dietary supplement) and exerts beneficial effects on female reproductive function. However, the actions of PAE on diminished ovarian reserve (DOR, a very common ovarian function disorder) are still unclear. Herein, our study investigated the effect and potential mechanism of PAE on DOR by using cisplatin-induced DOR mice and functional impairment of estradiol (E2) synthesis of ovarian granulosa-like KGN cells. Our data show that PAE improved the estrous cycle, ovarian index, and serum hormones levels, including E2, and the number of antral follicles and corpora lutea in DOR mice. Further mechanism results reveal that PAE promoted aromatase expression (the key rate-limiting enzyme for E2 synthesis) and upregulated the FSHR/cAMP/PKA/CREB signaling pathway in the ovaries. Subsequently, PAE improved the levels of E2 and aromatase and activated the FSHR/cAMP/PKA/CREB signaling pathway in KGN cells, while these improving actions were inhibited by the siRNA-FSHR and FSHR antagonist treatments. In sum, PAE restored the function of E2 synthesis in ovarian granulosa cells to improve DOR by activating the FSHR/cAMP/PKA/CREB signaling pathway, which exhibited a new clue for the development of effective therapeutic agents for the treatment of DOR.

Follicle-stimulating hormone receptor expression in advanced atherosclerotic plaques.

Experimental evidence indicates that follicle-stimulating hormone (FSH), an essential hormone for reproduction, can act directly on endothelial cells inducing atherosclerosis activation and development. However, it remains unknown whether the FSH-receptor (FSHR) is expressed in human atherosclerosis plaques. To demonstrate the FSHR presence, we used immunohistochemical and immunoelectron microscopy involving a specific monoclonal antibody FSHR1A02 that recognizes an epitope present in the FSHR-ectodomain. In all 55 patients with atherosclerotic plaques located in carotid, coronary, femoral arteries, and iliac aneurysm, FSHR was selectively expressed in arterial endothelium covering atherosclerotic plaques and endothelia lining intraplaque neovessels. Lymphatic neovessels were negative for FSHR. M1-macrophages, foam cells, and giant multinucleated cells were also FSHR-positive. FSHR was not detected in normal internal thoracic artery. Immunoelectron microscopy performed in ApoEKO/hFSHRKI mice with atherosclerotic plaques, after injection in vivo with mouse anti-hFSHR monoclonal antibody FSHR1A02 coupled to colloidal gold, showed FSHR presence on the luminal surface of arterial endothelial cells covering atherosclerotic plaques. Therefore, FSHR can bind, internalize, and deliver into the plaque circulating ligands to FSHR-positive cells. In conclusion, we report FSHR expression in endothelial cells, M1-macrophages, M1-derived foam cells, giant multinucleated macrophages, and osteoclasts associated with human atherosclerotic plaques.

Application of Biomarkers in Obese Infertile Women: A Genetic Tool for a Personalized Treatment.

This study investigates links between CART and leptin gene expression, FSH receptor Asn680Ser polymorphism, and reproductive hormones in morbidly obese patients under 40 years old, facing infertility, and undergoing bariatric surgery. A total of 29 women were included in this study. A hormonal profile along with detection of CART and leptin gene expression was evaluated before and after bariatric surgery. Additionally, the presence or absence of Asn680Ser of the FSHR gene was studied. Following bariatric surgery, a mean reduction in BMI (16.03 kg/m2) was observed in all women. FSH levels preoperatively varied significantly among genotypes, with medians of 8.1, 9.5, and 10.3 for individuals without polymorphism, heterozygotes, and homozygotes, respectively (p = 0.0408). Post surgery, marginal differences in FSH levels were observed (5.8, 7.1, and 8.2, respectively) (p = 0.0356). E2 and LH levels exhibited no significant genotype-based differences pre and post surgery. Presurgical E2 levels were 29.6, 29.8, and 29.6, respectively (p = 0.91634), while postsurgical levels were 51.2, 47.8, and 47 (p = 0.7720). LH levels followed similar patterns. Our findings highlight bariatric surgery's positive impact on BMI reduction and its potential connection to genetic markers, hormones, and infertility. This suggests personalized treatments and offers a valuable genetic tool for better fertility outcomes in obese individuals.

Effects of Vaccination against Recombinant FSH or LH Receptor Subunits on Gonadal Development and Functioning Male Rats.

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) play key roles in regulating testosterone secretion and spermatogenesis in male mammals, respectively, and they maintain the fertility of male animals by binding to their corresponding receptors. We designed and prepared a recombinant LH receptor (LHR) subunit vaccine and a recombinant FSH receptor (FSHR) subunit vaccine and used male Sprague Dawley (SD) rats as a model to examine their effects on testicular development, spermatogenesis, and testosterone secretion in prepubertal and pubertal mammals. Both vaccines (LHR-DTT and FSHR-DTT) significantly decreased the serum testosterone level in prepubertal rats (p < 0.05) but had no effect on the testosterone secretion in pubertal rats; both vaccines decreased the number of cell layers in the seminiferous tubules and reduced spermatogenesis in prepubertal and pubertal rats. Subunit vaccine FSHR-DTT decreased the sperm density in the epididymis in both prepubertal and pubertal rats (p < 0.01) and lowered testicular index and sperm motility in pubertal rats (p < 0.05), whereas LHR-DTT only reduced the sperm density in the epididymis in pubertal rats (p < 0.05). These results indicate that the FSHR subunit vaccine may be a promising approach for immunocastration, but it still needs improvements in effectiveness.

Honghua Xiaoyao tablet combined with estradiol improves ovarian function in D-galactose-induced aging mice by reducing apoptosis and affecting the release of reproductive hormones: an in vivo study.

Context: It is very necessary to delay ovarian aging and prevent age-related health problems. The active ingredient in Honghua Xiaoyao tablet (HHXYT) has the effects of anti-oxidation, anti-inflammation, immune regulation and so on. Objective: To explore the effect and mechanism of Honghua Xiaoyao tablet on aging model mice. Materials and methods: The aging model was established by intraperitoneal injection of D-galactose in model mice. The mice in the HHXYT-L,M,H group were given 0.3 g/kg, 0.6 g/kg and 1.2 g/kg Honghua Xiaoyao tablet suspension respectively, and the HHXYT-M + E2 group was given 0.6 g/kg HHXYT +0.13 mg/kg estradiol valerate for 30 days. In this study, ELISA, HE, Western blot, IH and TUNEL were used. Results: HHXYT + E2 can improve the gonadal index, estrous cycle of aging mice. In HHXYT-M + E2 group, the level of FSH and LH decreased, while E2 and AMH increased significantly. The number of growing follicles in HHXYT-M + E2 group increased, which was better than that of HHXYT alone. Western blot results showed that HHXYT-M + E2 group decreased the expression of Bax, cleaved-Parp, cleaved-Casp-3 and CytC molecules and increased the expression of Bcl-2 in ovarian tissue. FSHR expression decreased in model group and increased in HHXYT group. TUNEL staining showed that the number of apoptotic cells in HHXYT group was reduced, and the HHXYT-M + E2 group was the most significantly. Discussion and conclusion: HHXYT can improve the level of sex hormones and increase the number of growing follicles in aging mice. HHXYT-M + E2 group has the best effect, and its mechanism may be related to reducing ovarian granulosa cell apoptosis.

Puberty classifications in beef heifers are moderately to highly heritable and associated with candidate genes related to cyclicity and timing of puberty.

Introduction: Pubertal attainment is critical to reproductive longevity in heifers. Previously, four heifer pubertal classifications were identified according to attainment of blood plasma progesterone concentrations > 1 ng/ml: 1) Early; 2) Typical; 3) Start-Stop; and 4) Non-Cycling. Early and Typical heifers initiated and maintained cyclicity, Start-Stop started and then stopped cyclicity and Non-Cycling never initiated cyclicity. Start-Stop heifers segregated into Start-Stop-Discontinuous (SSD) or Start-Stop-Start (SSS), with SSD having similar phenotypes to Non-Cycling and SSS to Typical heifers. We hypothesized that these pubertal classifications are heritable, and loci associated with pubertal classifications could be identified by genome wide association studies (GWAS). Methods: Heifers (n = 532; 2017 - 2022) genotyped on the Illumina Bovine SNP50 v2 or GGP Bovine 100K SNP panels were used for variant component estimation and GWAS. Heritability was estimated using a univariate Bayesian animal model. Results: When considering pubertal classifications: Early, Typical, SSS, SSD, and Non-Cycling, pubertal class was moderately heritable (0.38 ± 0.08). However, when heifers who initiated and maintained cyclicity were compared to those that did not cycle (Early+Typical vs. SSD+Non-Cycling) heritability was greater (0.59 ± 0.19). A GWAS did not identify single nucleotide polymorphisms (SNPs) significantly associated with pubertal classifications, indicating puberty is a polygenic trait. A candidate gene approach was used, which fitted SNPs within or nearby a set of 71 candidate genes previously associated with puberty, PCOS, cyclicity, regulation of hormone secretion, signal transduction, and methylation. Eight genes/regions were associated with pubertal classifications, and twenty-two genes/regions were associated with whether puberty was attained during the trial. Additionally, whole genome sequencing (WGS) data on 33 heifers were aligned to the reference genome (ARS-UCD1.2) to identify variants in FSHR, a gene critical to pubertal attainment. Fisher's exact test determined if FSHR SNPs segregated by pubertal classification. Two FSHR SNPs that were not on the bovine SNP panel were selected for additional genotyping and analysis, and one was associated with pubertal classifications and whether they cycled during the trial. Discussion: In summary, these pubertal classifications are moderately to highly heritable and polygenic. Consequently, genomic tools to inform selection/management of replacement heifers would be useful if informed by SNPs associated with cyclicity and early pubertal attainment.

DENND1A desensitizes granulosa cells to FSH by arresting intracellular FSHR transportation.

Polycystic ovary syndrome (PCOS) is a complex disorder. Genome-wide association studies (GWAS) have identified several genes associated with this condition, including DENND1A. DENND1A encodes a clathrin-binding protein that functions as a guanine nucleotide exchange factor involved in vesicular transport. However, the specific role of DENND1A in reproductive hormone abnormalities and follicle development disorders in PCOS remain poorly understood. In this study, we investigated DENND1A expression in ovarian granulosa cells (GCs) from PCOS patients and its correlation with hormones. Our results revealed an upregulation of DENND1A expression in GCs from PCOS cases, which was positively correlated with testosterone levels. To further explore the functional implications of DENND1A, we generated a transgenic mouse model overexpressing Dennd1a (TG mice). These TG mice exhibited subfertility, irregular estrous cycles, and increased testosterone production following PMSG stimulation. Additionally, the TG mice displayed diminished responsiveness to FSH, characterized by smaller ovary size, less well-developed follicles, and abnormal expressions of FSH-priming genes. Mechanistically, we found that Dennd1a overexpression disrupted the intracellular trafficking of follicle stimulating hormone receptor (FSHR), promoting its internalization and inhibiting recycling. These findings shed light on the reproductive role of DENND1A and uncover the underlying mechanisms, thereby contributing valuable insights into the pathogenesis of PCOS and providing potential avenues for drug design in PCOS treatment.

Association of FSHR gene polymorphisms with poor ovarian response in patients undergoing IVF: A systematic review and meta-analysis.

BACKGROUND: The results of studies on the association between polymorphisms in the FSHR gene and the risk of POR undergoing IVF have been inconsistent with each other, so we conducted a meta-analysis of all the available studies to explore the association between polymorphisms in the FSHR gene and the risk of POR. METHODS: Literature that met the inclusion criteria was collected by searching six electronic databases and basic data from included studies were extracted. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the strength of association between follicle-stimulating hormone receptor (FSHR) gene polymorphism and poor ovarian response (POR) risk. Begg's and Egger's tests were used to determine whether there was publication bias, and sensitivity analysis and TSA analysis were used to verify the stability and reliability of the results. RESULTS: We included 24 articles, 22 of which explored rs6166, including 2,206 cases and 3,897 controls. 6 articles explored rs6165, including 444 cases and 875 controls. Under additive, heterozygote, and dominant models, rs6166 was significantly associated with POR (S vs. N: OR = 1.29, 95 % CI = 1.05-1.59, P = 0.017; NS vs. NN: OR = 1.33, 95 % CI = 1.02-1.74, P = 0.038; NS + SS vs. NN: OR = 1.38, 95 % CI = 1.04-1.84, P = 0.025). In ethnicity-based subgroup analyses, the additive, homozygote, heterozygote, and dominant models increased Asian POR risk. Among the five genetic models, rs6165 was significantly associated with POR (T vs. C: OR = 1.64, 95 % CI = 1.25-2.16, P = 0.000; TT vs. CC: OR = 2.76, 95 % CI = 1.43-5.32, P = 0.003; CT vs. CC: OR = 1.58, 95 % CI = 1.19-2.10, P = 0.001; TT vs. CC + CT: OR = 2.32, 95 % CI = 1.67-3.23, P = 0.000; CT + TT vs. CC: OR = 1.80, 95 % CI = 1.22-2.65, P = 0.003). In ethnicity-based subgroup analyses, all five genetic models increased the risk of POR in Caucasians. CONCLUSION: According to the current meta-analysis, the rs6166 S allele was significantly associated with an increased risk of POR, especially in Asian populations. The rs6165 T allele was significantly associated with an increased risk of POR, especially in Caucasian populations.

Follicle-Stimulating Hormone Receptor (FSHR) Ser680Asn Genotype Does Not Affect the Follicular Fluid Hormonal Profile in Stimulated Cycles Using Different Gonadotropin Preparations for Ovulation Induction: A Pilot Study.

BACKGROUND: The existing literature lacks consensus on the effectiveness of utilizing polymorphisms to enhance outcomes in in vitro fertilization (IVF), particularly regarding ovulation induction protocols, oocyte and embryo quality, and pregnancy rates. Therefore, the present pilot study aims to assess whether the composition of different gonadotropin preparations affects the ovarian stimulation protocol concerning follicle-stimulating hormone receptor (FSHR) Ser680Asn genotypes (Ser/Ser, Ser/Asn, and Asn/Asn), in terms of ovulation induction parameters, including oocyte maturation rate, embryo quality, and pregnancy rate. METHODOLOGY: A total of 94 IVF patients underwent treatment using a GnRH antagonist protocol with four distinct gonadotropin preparations: HMG, HMG/hCG, rFSH, and rFSH/hCG. Follicular fluid (FF) samples were pooled for each patient for analysis. RESULTS: No statistical differences in the FF hormonal profile (progesterone, testosterone, androstenedione, estradiol, FSH, hCG) among the FSHR genotypes were reported either separately for each protocol or in combination for the four different preparations of gonadotropins. The maturation rate of MII oocytes and embryo quality did not differ among women carrying either Ser/Ser, Ser/Asn, or Asn/Asn genotype (p-value=0.475, and p-value=1.000, respectively). Moreover, no statistically significant correlation was revealed among Ser/Ser, Ser/Asn, and Asn/Asn carriers and pregnancy rate (p = 0.588). CONCLUSIONS: FF hormonal analysis of women undergoing IVF using different ovulation induction protocols and carrying either Ser/Ser, Ser/Asn, or Asn/Asn genotype revealed no significant correlations, in terms of maturation rate of MII oocytes, embryo quality, and pregnancy rate, indicating that the FSHR Ser680Asn genotype does not constitute a biomarker for a positive pregnancy outcome. Therefore, the existence of a different mechanism for the expression of FSHR Ser680Asn genotypes in the FF hormonal profile related to stimulated cycles is implied.

Core fucosylation regulates the ovarian response via FSH receptor during follicular development.

INTRODUCTION: Ovarian low response to follicle-stimulating hormone (FSH) causes infertility featuring hypergonadotropic hypogonadism, ovarian failure, and/or defective ovarian response. OBJECTIVES: N-glycosylation is essential for FSH receptor (FSHR). Core fucosylation catalyzed by fucosyltransferase 8 (FUT8) is the most common N-glycosylation. Core fucosylation level changes between individuals and plays important roles in multiple physiological and pathological conditions. This study aims to elucidate the significance of FUT8 to modulate FSHR function in female fertility. METHODS: Samples from patients classified as poor ovary responders (PORs) were detected with lectin blot and real-time PCR. Fut8 gene knockout (Fut8-/-) mice and FUT8-knockdown human granulosa cell line (KGN-KD) were established and in vitro fertilization (IVF) assay, western blot, molecular interaction, immunofluorescence and immunoprecipitation were applied. RESULTS: Core fucosylation is indispensable for oocyte and follicular development. FSHR is a highly core-fucosylated glycoprotein. Loss of core fucosylation suppressed binding of FSHR to FSH, and attenuated FSHR downstream signaling in granulosa cells. Transcriptomic analysis revealed the downregulation of several transcripts crucial for oocyte meiotic progression and preimplantation development in Fut8-/- mice and in POR patients. Furthermore, loss of FUT8 inhibited the interaction between granulosa cells and oocytes, reduced transzonal projection (TZP) formation and caused poor developmental competence of oocytes after fertilization in vitro. While L-fucose administration increased the core fucosylation of FSHR, and its sensitivity to FSH. CONCLUSION: This study first reveals a significant presence of core fucosylation in female fertility control. Decreased fucosylation on FSHR reduces the interaction of FSH-FSHR and subsequent signaling, which is a feature of the POR patients. Our results suggest that core fucosylation controls oocyte and follicular development via the FSH/FSHR pathway and is essential for female fertility in mammals.

Peroxiredoxin 4 deficiency induces accelerated ovarian aging through destroyed proteostasis in granulosa cells.

Ovarian aging, a complex and challenging concern within the realm of reproductive medicine, is associated with reduced fertility, menopausal symptoms and long-term health risks. Our previous investigation revealed a correlation between Peroxiredoxin 4 (PRDX4) and human ovarian aging. The purpose of this research was to substantiate the protective role of PRDX4 against ovarian aging and elucidate the underlying molecular mechanism in mice. In this study, a Prdx4-/- mouse model was established and it was observed that the deficiency of PRDX4 led to only an accelerated decline of ovarian function in comparison to wild-type (WT) mice. The impaired ovarian function observed in this study can be attributed to an imbalance in protein homeostasis, an exacerbation of endoplasmic reticulum stress (ER stress), and ultimately an increase in apoptosis of granulosa cells. Furthermore, our research reveals a noteworthy decline in the expression of Follicle-stimulating hormone receptor (FSHR) in aging Prdx4-/- mice, especially the functional trimer, due to impaired disulfide bond formation. Contrarily, the overexpression of PRDX4 facilitated the maintenance of protein homeostasis, mitigated ER stress, and consequently elevated E2 levels in a simulated KGN cell aging model. Additionally, the overexpression of PRDX4 restored the expression of the correct spatial conformation of FSHR, the functional trimer. In summary, our research reveals the significant contribution of PRDX4 in delaying ovarian aging, presenting a novel and promising therapeutic target for ovarian aging from the perspective of endoplasmic reticulum protein homeostasis.

The Effect of FSHR (G2039A) Polymorphism on Müllerian Duct Development and Hormonal Profile of Women with Primary Amenorrhea.

Background: The function of follicle-stimulating hormone (FSH) is mediated by binding to its G-protein coupled receptor (GPCR) which is expressed on granulosa cells of the ovary. The purpose of the current study was to examine the impact of FSHR G2039A polymorphism (rs6166; Ser680Asn) on clinical and radiology profiles of women with primary amenorrhea (PA) in Gujarat, India. Methods: A total of 90 women (45 controls and 45 cases) were recruited for the study after obtaining informed consent. The DNA extraction was performed on the venous blood samples collected from the participants, followed by polymerase chain reaction (PCR). The presence of polymorphism was then analyzed using restriction fragment polymorphism (RFLP) with the BSeNI enzyme. The statistical analysis was conducted using an independent t-test, chi-square test, and ANOVA. Significance was determined by a p<0.05. Results: Results revealed that homozygous wild type genotype was observed in 46.7% (n=21) of the control group and 11.1% (n=5) of the case group. Heterozygous genotype was observed in 33.3% (n=15) of the control group and 55.6% (n=25) of the case group (p<0.001). Homozygous mutant genotype was observed in 20% (n=9) of the control group and 33.3% (n=15) of the case group (p<0.01). The hormonal profile revealed that serum levels of FSH and luteinizing hormone (LH) were significantly higher (p<0.05) in the AA and AG genotypes compared to the GG genotypes. Conclusion: The FSHR rs6166 G2039A was associated with PA in women, and the A allele could be considered a causative risk factor in developing the condition.

Diferencias en los polimorfismos de genes asociados al control del desarrollo folicular entre búfalos (Bubalus bubalis) y vacunos (Bos indicus) / Differences in polymorphisms of genes associated with follicle development control between buffaloes (Bubalus bubalis) and cattle (Bos indicus) / Diferenças nos polimorfismos genéticos associados ao controle do desenvolvimento folicular entre búfalos (Bubalus bubalis) e bovinos (Bos indicus)

Resumen Se han encontrado asociaciones entre polimorfismos genéticos y parámetros reproductivos, este abordaje adicionalmente permitiría proponer explicaciones a diferentes fenómenos estudiados. FSHr, InhA, AMH y AMHr son genes asociados al desarrollo folicular, que tienen una alta homología y la misma función en bovinos. El objetivo del presente trabajo fue evaluar en bovinos muy relacionados si se encuentran algunos polimorfismos asociados a parámetros reproductivos reportados en otras especies. Durante el 2018, en el Magdalena Medio Colombiano fueron tomadas muestras de sangre anticoagulada de 50 búfalas mestizas (Bubalus bubalis) y 50 vacas cebú comercial (Bos indicus). Se registró la paridad, días abiertos, intervalo parto primer servicio y edad al primer parto de cada animal, sin alteraciones anatómicas, con edad y peso similar, mantenidos en las mismas condiciones de alimentación y manejo. Se extrajo el ADN por el método de Salting out y se evaluaron los polimorfismos de acuerdo con lo reportado en la literatura. Se compararon los datos usando la prueba Mann Whitney, se consideró significativo un valor de p<0,05. Se encontró que los búfalos tienen menores niveles de AMH, edad al primer parto, intervalo ente partos que las vacas (p<0,001), mayor paridad (p=0,005). No se confirmaron los po- limorfismos reportados en Holstein y en humanos en ninguna de las muestras y especies analizadas. No confirmar los hallazgos en bovinos de otras razas y espe- cies, aunque las especies tengan diferentes parámetros reproductivos, muestra la necesidad de replantear el abordaje del estudio de la asociación de los fenómenos reproductivos y genéticos.

Abstract Associations have been found between genetic polymorphisms and reproductive parameters, this approach additionally would allow us to propose explanations to the phenomena studied. FSHr, InhA, AMH and AMHr are genes associated with follicular development, which have a high homology and the same function in bovines. The objective of this work was to evaluate if some polymorphisms described in other species associated with reproductive parameters are found in two closely related bovines. During 2018, in the Colombian Magdalena Medio anticoagulated blood samples from 50 buffaloes (Bubalus bubalis) and 50 commercial zebu cows (Bos indicus) were taken. All animals without anatomical abnormalities and similar weight and age were taken and the. parity, intercalving period and age at first calving were also recorded. DNA were extracted using Salting out method and the reported polymorphisms of the above mentioned genes were evaluated. Data were compared using Mann Whitney test and p<0.05 value were considered significant. Buffaloes have lower AMH levels, age at first calving, calving interval than Bos indicus cows (p<0.001) and higher parity (p=0.005). The polymorphisms reported in Holstein and humans were not confirmed in any of the samples and species analyzed. The results, are in some way paradoxical because there are differences in reproductive parameters but nothing different in the studies genes. It shows the need to rethink the approach of the study of the association of reproductive and genetic phenomena

Resumo Foram encontradas associações entre polimorfismos genéticos e parâmetros repro- dutivos; essa abordagem também permitiria propor explicações para os diferentes fenômenos estudados. FSHr, InhA, AMH e AMHr são genes associados ao desenvol- vimento folicular, que apresentam alta homologia e a mesma função em bovinos. O objetivo do presente trabalho foi avaliar em bovinos intimamente relacionados se alguns polimorfismos associados a parâmetros reprodutivos relatados em outras espécies. Em 2018, 50 vacas de búfalo (Bubalus bubalis) e 50 de zebu comerciais (Bos indicus) foram amostradas na Magdalena Medio colombiana. Foram registra- das paridade, dias abertos, intervalo de nascimento do primeiro serviço e idade no primeiro nascimento de cada animal, sem alterações anatômicas, com idade e peso semelhantes, mantidas nas mesmas condições de alimentação e manuseio. O DNA foi extraído pelo método Salting out e os polimorfismos foram avaliados de acordo com o relatado na literatura. Os dados foram comparados pelo teste de Mann Whitney, sendo considerado significativo um valor de p <0,05. Verificou-se que os búfalos apresentam níveis mais baixos de AMH, idade do primeiro nascimento, intervalo entre os nascimentos que as vacas (p <0,001), maior paridade (p = 0,005). Os polimorfis- mos relatados em Holstein e em humanos não foram confirmados em nenhuma das amostras e espécies analisadas. A não confirmação dos achados em bovinos de outras raças e espécies, embora as espécies possuam diferentes parâmetros repro- dutivos, mostra a necessidade de repensar a abordagem do estudo da associação dos fenômenos reprodutivos e genéticos.

Resultados en ciclos de FIV-ICSI adicionando hormona luteinizante recombinante a la hormona folículo estimulante recombinante con menotropinas durante la estimulación ovárica en pacientes mayores de 35 años / Results in IVF-ICSI cycles adding luteinizing hormone recombinant to follicle stimulating hormone recombinant with menotropins during ovarian stimulation in patients over 35 years-old

Resumen OBJETIVO Evaluar los resultados en ciclos de FIV-ICSI de dos protocolos de estimulación ovárica en mujeres mayores de 35 años e investigar si agregar hormona luteinizante recombinante a FSH-r en un protocolo de estimulación mejora la respuesta ovárica y, en consecuencia, las tasas de embarazo en este grupo poblacional. MATERIALES Y MÉTODOS Estudio longitudinal, observacional y retrospectivo efectuado en pacientes de la Clínica de Reproducción Hisparep del Hospital Español con diagnóstico de infertilidad, mayores de 35 años, que recibieron un ciclo de hiperestimulación ovárica controlada con FIV-ICSI durante el periodo 2014-2016. El análisis estadístico se efectuó con la prueba de t de Student para muestras independientes. Los estudios se analizaron con el paquete estadístico SPSS IBM, versión 22. RESULTADOS Se analizaron 201 mujeres con infertilidad, mayores de 35 años. El grupo 1 (n = 101) de FIV-ICSI recibió estimulación con hormona folículo estimulante recombinante y hormona luteinizante recombinante 2:1 con menotropinas (Pergoveris® y Merapur®) a partir del segundo día del ciclo. El grupo 2 (n = 100) recibió hormona folículo estimulante recombinante y menotropinas (Gonal F® y Merapur®); en ambos esquemas se utilizó antagonista de GnRH a partir del día 7 del ciclo. La media de ovocitos aspirados fue de 7.5 en el grupo 1 y 9.1 en el grupo 2 (p = 0.058). La media de ovocitos maduros fue 6.2 en el Grupo 1 vs 7.4 en el grupo 2 (p = 0.085). La tasa de fecundación en el grupo 1 fue de 57 vs 67% en el grupo 2 (p = 0.045). En el grupo 1 la tasa de implantación por embrión transferido en fresco fue 24.1 vs 10.3% en el Grupo 2 (p = 0.40), la tasa de recién nacido vivo fue de 30% en el Grupo 1 vs 20.6% en el Grupo 2. La media de embriones vitrificados en el Grupo 1 fue 1.47 vs 1.38 en el Grupo 2. CONCLUSIONES La probable ventaja de la complementación con hormona folículo estimulante recombinante durante la estimulación ovárica en mujeres mayores de 35 años es de interés y se requiere su evaluación en estudios posteriores.

Abstract OBJECTIVE To evaluate the reproductive effects when recombinant luteinizing hormone is added and to compare two stimulation schemes by number of aspirated oocytes, mature oocytes, fertilization and implantation rates, live newborn and number of vitrified embryos. MATERIALS AND METHODS Longitudinal, observational and retrospective study carried out in patients of the Hisparep Reproduction Clinic of the Spanish Hospital with diagnosis of infertility, over 35 years old, who received a controlled ovarian hyperstimulation cycle with IVF-ICSI during the period 2014-2016. The statistical analysis was carried out with the Student t test for independent samples. The studies were analyzed with the IBM SPSS statistical package, version 22. RESULTS We analyzed 201 women with infertility, over 35 years of age. Group 1 (n = 101) of IVF-ICSI received stimulation with recombinant follicle-stimulating hormone and recombinant luteinizing hormone 2: 1 with menotropins (Pergoveris® and Merapur®) from the second day of the cycle. Group 2 (n = 100) received recombinant follicle stimulating hormone and menotropins (Gonal F® and Merapur®); in both schemes, GnRH antagonist was used from day 7 of the cycle. The average number of aspirated oocytes was 7.5 in Group 1 and 9.1 in Group 2 (p = 0.058). Mean mature oocytes were 6.2 in Group 1 vs 7.4 in Group 2 (p = 0.085). The fertilization rate in group 1 was 57 vs. 67% in Group 2 (p = 0.045). In Group 1 the implantation rate per embryo transferred fresh was 24.1 vs 10.3% in Group 2 (p = 0.40), the live newborn rate was 30% in Group 1 vs 20.6% in Group 2. The mean number of vitrified embryos in Group 1 was 1.47 vs 1.38 in Group 2. CONCLUSIONS The probable advantage of supplementation with recombinant follicle-stimulating hormone during ovarian stimulation in women over 35 years of age is of interest and its evaluation is required in subsequent studies.

Phytohemagglutinin improves the development and ultrastructure of in vitro-cultured goat (Capra hircus) preantral follicles

The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles cultured in vitro. Secondary follicles (∼0.2 mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in α-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200 µg/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [α-MEM+ (94.59%); 1 µg/mL PHA (96.43%); 10 µg/mL PHA (84.85%); 50 µg/mL PHA (85.29%); 100 µg/mL PHA (88.57%), and 200 µg/mL PHA (87.50)], but the presence of 10 µg/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 ± 0.1) and PCNA (4.4 ± 0.2) was also significantly increased in follicles cultured with 10 µg/mL PHA in relation to those cultured in α-MEM+ (1.0 ± 0.1). In conclusion, supplementation of culture medium with 10 µg/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.

Goat ovarian follicles express different levels of mRNA for inhibin-ßA subunit and activin-A stimulates secondary follicle growth in vitro / Folículos ovarianos caprinos expressam diferentes níveis de RNAm para subunidade ßA da inibina e ativina-A promove o crescimento de folículos secundários in vitro

This study investigated the levels of messenger ribonucleic acids (mRNA) for inhibin-ßA subunit in goat primordial, primary and secondary follicles, as well as in cumulus-oocyte complexes (COCs) and mural granulosa / theca cells of antral follicles. The effects of activin-A (100ng mL-1) and/or follicle stimulating hormone (FSH, 50ng mL-1) on growth and expression of mRNA for activin-A and FSH receptor (FSH-R) in secondary follicles cultured for six days were evaluated. The data showed that the expression of inhibin-ßA is lower in secondary follicles than in primary follicles and is higher in large antral follicles than in small antral follicles. After culture, activin-A and/or FSH promoted growth of secondary follicles, while FSH increased the levels of mRNA for inhibin-ßA, and activin-A increased the levels of FSH-R mRNA. In conclusion, mRNA for inhibin-ßA is expressed at different levels in pre-antral and antral follicles and activin-A acts as a stimulator of the FSH-R expression in goat follicles. On its turn, the expression of inhibin-ßA is stimulated by FSH, which together with activin-A promotes secondary follicle growth in-vitro.

Este estudo investigou os níveis de ácidos ribonucleicos (RNAm) para a subunidade ßA da inibina em folículos primordiais, primários e secundários caprinos, bem como em complexos cumulus-oócitos (CCOs) e células da granulosa mural/teca de folículos antrais. Além disso, avaliaram-se os efeitos da ativina-A (100ng mL-1) e/ou hormônio folículo estimulante (FSH, 50ng mL-1) sobre o crescimento e a expressão do RNAm para inibina-ßA e receptores de FSH (FSH-R) em folículos secundários cultivados por seis dias. Os dados mostraram que a expressão de inibina-ßA é menor em folículos secundários do que em folículos primários e é maior em grandes folículos antrais que nos pequenos folículos antrais. Após o cultivo, ativina-A e/ou FSH promoveram o crescimento de folículos secundários. Enquanto o FSH aumentou os níveis de RNAm para inibina-ßA, a ativina-A aumentou os níveis de RNAm para FSH-R. Em conclusão, a inibina-ßA é expressa em diferentes níveis em folículos pré-antrais e antrais e a ativina-A atua como um estimulador da expressão de FSH-R em folículos caprinos. Por sua vez, a expressão de inibina-ßA é estimulada pelo FSH, que, juntamente com ativina, promove o crescimento de folículos secundários in vitro.