Basal FSH values are positively associated with aneuploidy incidence in pre-advanced maternal age (AMA) but not in AMA patients.

PURPOSE: To assess the impact of maternal age on the association between maternal basal FSH and aneuploidy. METHODS: A retrospective study including data from 1749 blastocysts diagnosed as euploid or aneuploid by PGT-A (preimplantation genetic testing for aneuploidy). Aneuploidy incidence was compared between embryos from mothers with high vs. low basal FSH levels (above and below the group median, respectively) in total, pre-AMA (advanced maternal age; < 35 years, 198 embryos) and AMA (≥ 35 years, 1551 embryos) patient groups, separately. To control for the interference of potentially confounding variables, the association between aneuploidy and high basal FSH levels was assessed by multivariate logistic analysis in overall, pre-AMA and AMA patient groups. RESULTS: Overall, aneuploidy rate was 9% higher (p = 0.02) in embryos from patients with high basal FSH (63.7%) compared to those with low basal FSH (58.4%). In the pre-AMA subgroup, aneuploidy incidence was 35% higher (p = 0.04) in embryos from patients with high basal FSH (53.5%) compared to those with low basal FSH (39.4%). Differently, aneuploidy occurrence did not vary between embryos from AMA patients with low (61.0%) and high (64.8%) basal FSH (p = 0.12). The multivariate analysis revealed that, in pre-AMA embryos, the association between aneuploidy occurrence and high basal FSH is independent of potential confounding variables (p = 0.04). CONCLUSION: Maternal basal FSH values are associated with embryo aneuploidy in pre-AMA but not in AMA patients. The present findings suggest that basal FSH is a useful parameter to assess aneuploidy risk in pre-AMA patients and reinforce the hypothesis that excessive FSH signalling can predispose to oocyte meiotic errors.

The impact of oocyte central granularity on ICSI practice: developmental competence of dysmorphic and morphologically normal companion oocytes.

PURPOSE: To assess the effects of oocyte central granularity and its underlying endocrine environment on developmental competence of dysmorphic and morphologically normal oocytes. METHODS: Retrospective cohort study including 1,082 patients undergoing autologous ICSI cycles. Of these, 211 patients provided 602 oocytes with central granularity (CG) and 427 morphologically normal cycle companion oocytes (NCG). The remaining 871 patients provided only morphologically normal oocytes in cycles not yielding dysmorphic oocytes (N). Patient profile associated with CG was characterized, and fertilization rates, early morphokinetics and live birth rates were compared between N, CG and NCG groups. Patient characteristics associated with implantation and delivery performance of CG-derived embryos were assessed. RESULTS: CG was associated with higher maternal age, basal FSH concentrations and total FSH dose, but with lower circulating AMH (p ≤ 0.035). Fertilization rates were reduced and early morphokinetic parameters were delayed in CG (p < 0.025) and NCG (p < 0.05) groups as compared to the N group. Embryos derived from CG oocytes achieved a markedly lower live birth rate (14.9%) as compared to those derived from NCG (36.8%; p = 0.03) and N oocytes (29.8%; p = 0.002). The negative relationship between CG and live birth was confirmed by a multivariate analysis controlling for potential confounders (OR:2.59, IC:1.27-5.31; P = 0.009). Implantation and delivery rates following transfers of CG-derived embryos were inversely associated with maternal age. CONCLUSION: CG oocytes, but not their morphologically normal cycle companions, have severely compromised developmental competence. Maternal age should be a key parameter in deciding whether or not to utilize CG oocytes in ICSI cycles.

Oocyte secreted factors control genes regulating FSH signaling and the maturation cascade in cumulus cells: the oocyte is not in a hurry.

PURPOSE: To assess the effects of the oocyte on mRNA abundance of FSHR, AMH and major genes of the maturation cascade (AREG, EREG, ADAM17, EGFR, PTGS2, TNFAIP6, PTX3, and HAS2) in bovine cumulus cells. METHODS: (1) Intact cumulus-oocyte complexes, (2) microsurgically oocytectomized cumulus-oolema complexes (OOX), and (3) OOX + denuded oocytes (OOX+DO) were subjected to in vitro maturation (IVM) stimulated with FSH for 22 h or with AREG for 4 and 22 h. After IVM, cumulus cells were separated and relative mRNA abundance was measured by RT-qPCR. RESULTS: After 22 h of FSH-stimulated IVM, oocytectomy increased FSHR mRNA levels (p=0.005) while decreasing those of AMH (p=0.0004). In parallel, oocytectomy increased mRNA abundance of AREG, EREG, ADAM17, PTGS2, TNFAIP6, and PTX3, while decreasing that of HAS2 (p<0.02). All these effects were abrogated in OOX+DO. Oocytectomy also reduced EGFR mRNA levels (p=0.009), which was not reverted in OOX+DO. The stimulatory effect of oocytectomy on AREG mRNA abundance (p=0.01) and its neutralization in OOX+DO was again observed after 4 h of AREG-stimulated IVM. After 22 h of AREG-stimulated IVM, oocytectomy and addition of DOs to OOX caused the same effects on gene expression observed after 22 h of FSH-stimulated IVM, except for ADAM17 (p<0.025). CONCLUSION: These findings suggest that oocyte-secreted factors inhibit FSH signaling and the expression of major genes of the maturation cascade in cumulus cells. These may be important actions of the oocyte favoring its communication with cumulus cells and preventing premature activation of the maturation cascade.

Perinatal outcomes in 13,626 singleton pregnancies after autologous IVF across three continents over 7 years.

PURPOSE: Are trends in singleton autologous IVF perinatal outcomes consistent over time among five international infertility centers? METHODS: This was a retrospective cohort study from January 1, 2012, to December 31, 2018. This study was performed through a large infertility network at five international infertility centers in which patients who had a singleton live birth resulting from fresh and frozen autologous IVF cycles were included. The primary outcome was live birth weight (BW) with secondary outcomes of preterm birth (PTB), large for gestational age (LGA), small for gestational age (SGA), and gestational age at delivery. RESULTS: The entire cohort (n = 13,626) consisted of 6941 fresh and 6685 frozen autologous IVF cycles leading to singleton deliveries. Maternal age, parity, body mass index, neonatal sex, and GA at delivery were similar for fresh and frozen IVF cycles in the entire cohort and within each infertility center. Four centers had a trend of decreased BW and three centers had decreased rates of PTB before 32 and 28 weeks and LGA newborns annually, although significance was not reached. Three IVF centers had annual increased trends of PTB before 37 weeks and four centers had increased rates of SGA newborns, although significance was not reached. CONCLUSION: Similar trends in perinatal outcomes were present across five international infertility centers over 7 years. Additional studies are crucial to further assess and optimize perinatal outcomes at an international level.

Perinatal outcomes in 6640 singleton pregnancies after donor oocyte IVF across three continents over 7 years.

PURPOSE: Are trends in singleton donor oocyte IVF perinatal outcomes consistent over time among four international ethnically diverse infertility centers? METHODS: This retrospective cohort consisted of an infertility network of four international IVF centers across three continents. Singleton live births resulting from fresh and frozen donor oocyte embryo transfers from January 1, 2012 to December 31, 2018 were included. The main outcome measures were birth weight (BW), preterm birth (PTB), large for gestational age (LGA), small for gestational age (SGA) and gestational age (GA) at delivery. RESULTS: The entire cohort (n = 6640) consisted of 4753 fresh and 1887 frozen donor oocyte embryo transfers. Maternal age, parity, body mass index, neonatal sex and GA at delivery were similar for fresh and frozen donor oocyte embryo transfers in the entire cohort and within each infertility center. All four centers had a trend of decreased BW and rates of PTB before 32 weeks annually, although significance was not reached. Three of the four centers had annual increased trends of PTB before 37 weeks and LGA newborns, although significance was not reached. BWs for the entire cohort for fresh and frozen donor embryo transfers were 3166 g ± 601 g and 3137 g ± 626 g, respectively. CONCLUSION: Similar trends in perinatal outcomes were present across four international infertility centers over 7 years. The overall perinatal trends in donor oocyte IVF may be applicable to centers worldwide, but further studies in more geographic regions are needed.

Maternal age affects the relationship of basal FSH and anti-Müllerian hormone concentrations with post-ICSI/IVF live birth.

RESEARCH QUESTION: Does the association of basal FSH and anti-Müllerian hormone (AMH) concentrations with post-IVF/intracytoplasmic sperm injection (ICSI) live birth change with maternal age? DESIGN: A total of 2003 IVF/ICSI patients were stratified according to basal FSH/AMH in concordant favourable (CF; AMH >1 ng/ml and FSH ≤10 IU/l), concordant unfavourable (CU; AMH ≤1 ng/ml and FSH >10 IU/l), discordant with favourable AMH (DFA) and discordant with favourable FSH (DFF) groups, as well as according to age in pre-advanced maternal age (pre-AMA; <35), AMA-1 (≥35, ≤37), AMA-2 (>37, ≤40) and AMA-3 (>40). IVF/ICSI outcomes were compared among CF, CU, DFA and DFF groups, and the association of basal FSH and AMH concentrations with live birth was tested by univariate and multivariate analysis in total, pre-AMA and AMA groups, separately. RESULTS: Different outcome patterns were observed in discordant AMH/FSH groups from different age categories; favourable basal FSH concentrations were associated with higher delivery rates in pre-AMA patients, but with lower delivery rates in AMA groups. Within pre-AMA patients, DFF patients presented higher delivery rates but lower oocyte yield compared with DFA patients. In the univariate analysis, favourable AMH (P < 0.02) and oocyte yield (P < 0.002) were positively associated with live birth in all AMA groups. The multivariate analysis revealed that favourable basal FSH, but not AMH or oocyte yield, is associated with live birth in pre-AMA patients independently of other variables (P = 0.012). CONCLUSIONS: The relationship of basal FSH and AMH with IVF/ICSI success changes with maternal age; basal FSH better reflects clinical outcomes probably determined by oocyte quality in pre-AMA patients, while AMH better suits AMA patients.

Physiological parameters related to oocyte nuclear differentiation for the improvement of IVM/IVF outcomes in women and cattle.

In vitro maturation (IVM) has been applied in numerous different contexts and strategies in humans and animals, but in both cases it represents a challenge still far from being overcome. Despite the large dataset produced over the last two decades on the mechanisms that govern antral follicular development and oocyte metabolism and differentiation, IVM outcomes are still unsatisfactory. This review specifically focuses on data concerning the potential consequences of using supraphysiological levels of FSH during IVM, as well as on the regulation of oocyte chromatin dynamics and its utility as a potential marker of oocyte developmental competence. Taken together, the data revisited herein indicate that a significant improvement in IVM efficacy may be provided by the integration of pre-OPU patient-specific protocols preparing the oocyte population for IVM and more physiological culture systems mimicking more precisely the follicular environment that would be experienced by the recovered oocytes until completion of metaphase II.

Early embryo morphokinetics is a better predictor of post-ICSI live birth than embryo morphology: speed is more important than beauty at the cleavage stage.

Given the importance of embryo developmental competence assessment in reproductive medicine and biology, the aim of this study was to compare the performance of fertilization and cleavage morphokinetics with embryo morphology to predict post-ICSI live birth. Data from embryos cultured in a time-lapse microscopy (TLM) incubator and with known live birth outcomes (LB: embryos achieving live birth, n = 168; NLB: embryos not achieving live birth, n = 1633) were used to generate receiver operating characteristic (ROC) curves based on morphokinetic or morphological scores, and the respective areas under the curve (AUC) were compared. The association between live birth and 12 combinations of four morphokinetic quality degrees (A-D) with three morphological quality degrees (A-C) was assessed using multivariate analysis. Morphokinetic parameters from tPNa to t8 were reached earlier in LB compared with NLB embryos. The ROC curve analysis indicated that morphokinetic information is more accurate than conventional morphology to predict live birth [AUC = 0.64 (95% CI 0.58-0.70) versus AUC = 0.58 (95% CI 0.51-0.65)]. The multivariate analysis was in line with AUCs, revealing that embryos with poor morphokinetics, independently of their morphology, provide lower live birth rates (P < 0.001). A considerable percentage of embryos with top morphology presented poor morphokinetics (20.10%), accompanied by a severely reduced live birth rate in comparison with embryos with top morphology and morphokinetics (P < 0.001). In conclusion, TLM-derived early morphokinetic parameters were more predictive of live-birth achievement following ICSI than conventional morphology.

Bone morphogenetic protein 15 supplementation enhances cumulus expansion, nuclear maturation and progesterone production of in vitro-matured bovine cumulus-oocyte complexes.

In vitro embryo production (IVP) efficiency is reduced when compared to in vivo. The basic knowledge of bovine in vitro oocyte maturation (IVM) mechanisms provides support to improve in vitro embryo production yields. The present study assessed the effects of bone morphogenetic protein 15 (BMP15), fibroblast growth factor 16 (FGF16) and their combined action on cumulus cells (CC) expansion, oocyte and CC DNA fragmentation, oocyte nuclear maturation, energetic metabolism and progesterone production in bovine IVM. Cumulus-oocyte complexes (COC) were matured in control or supplemented media containing BMP15 (100 ng/ml), FGF16 (10 ng/ml) or BMP15 combined with FGF16; and assessed at 0 and 22 hr of IVM. BMP15 alone or its association with FGF16 enhanced cumulus expansion. BMP15 decreased DNA fragmentation in both CC and oocytes, and improved oocyte nuclear maturation rate. In addition, BMP15 increased CC progesterone production, an effect not previously reported. The present study reinforces previous data pointing to a beneficial influence of BMP15 during IVM, while providing novel evidence that the underlying mechanisms involve increased progesterone production.

Fibroblast growth factor 2 regulates cumulus differentiation under the control of the oocyte.

PURPOSE: We first assessed regulation of FGF2 expression in cumulus cells by FSH and oocyte-secreted factors during in vitro maturation (IVM). Then, we tested the hypothesis that FGF2 regulates meiotic progression, cumulus expansion, and apoptosis in cumulus-oocyte complexes (COC) undergoing IVM. METHODS: In vitro maturation of bovine COC was utilized as a model to assess regulation of FGF2 expression by FSH and oocyte-secreted factors (via microsurgical removal of the oocyte), as well as effects of graded doses of FGF2 on meiotic progression, degree of cumulus expansion, dissociation of cumulus cells, and cumulus cells apoptosis. Expression of genes regulating functional endpoints altered by FGF2 treatment was assessed in cumulus cells by real-time PCR. Cultures were replicated 4-5 times and effects of treatments were tested by ANOVA. RESULTS: FGF2 mRNA expression was increased by FSH and oocyte-secreted factors during IVM. Addition of FGF2 to the IVM medium advanced meiosis resumption, decreased the ease with which cumulus cells were dissociated, and inhibited cumulus cells apoptosis. Decreased cumulus dissociation was accompanied by decreased expression of TNFAIP6. CONCLUSIONS: This is the first study showing that FGF2 expression is regulated by the oocyte in cumulus cells. Moreover, we report novel effects of FGF2 on cumulus cell survival and extracellular matrix (ECM) quality during IVM that may favor acquisition of developmental competence and suggest physiological roles during the final steps of COC differentiation.

Maternal body mass index affects embryo morphokinetics: a time-lapse study.

PURPOSE: To assess the effect of body mass index (BMI) on morphokinetic parameters of human embryos evaluated with time-lapse technology during in vitro culture. METHODS: A retrospective analysis of ART cycles utilizing time-lapse technology was undertaken to assess the potential impact of maternal BMI on morphokinetic and static morphological parameters of embryo development. The cohort of patients was divided into four groups: 593 embryos from 128 underweight women in group A; 5248 embryos from 1107 normal weight women in group B; 1053 embryos from 226 overweight women in group C; and 286 embryos from 67 obese women in group D. RESULTS: After adjusting for maternal age, paternal age, and cause of infertility, time to reach five blastomeres (t5) and time to reach eight blastomeres (t8) were longer in obese women compared with normoweight women [50.84 h (46.31-55.29) vs. 49.24 h (45.69-53.22) and 57.89 h (51.60-65.94) vs. 55.66 h (50.89-62.89), adjusted p < 0.05 and adjusted p < 0.01, respectively]. In addition, t8 was also delayed in overweight compared with normoweight women [56.72 h (51.83-63.92) vs. 55.66 h (50.89-62.89), adjusted p < 0.01]. No significant differences were observed among groups with regard to embryo morphology and pregnancy rate. Miscarriage rate was higher in underweight compared with normoweight women (OR = 2.1; 95% CI 1.12-3.95, adjusted p < 0.05). CONCLUSION: Assessment with time-lapse technology but not by classical static morphology evidences that maternal BMI affects embryo development. Maternal obesity and overweight are associated with slower embryo development.

Steroid hormones interact with natriuretic peptide C to delay nuclear maturation, to maintain oocyte-cumulus communication and to improve the quality of in vitro-produced embryos in cattle.

In vivo, oocyte maturation is triggered by the ovulatory LH surge, whereas in vitro it is precociously induced when the cumulus-oocyte complex is removed from the follicle. Natriuretic peptide C (NPPC) delays germinal vesicle breakdown (GVBD) while increasing oocyte-cumulus communication during in vitro maturation (IVM) in cattle. In the present study we first tested the hypothesis that steroids secreted by the follicle (17ß-oestradiol, progesterone and androstenedione) interact with NPPC to delay GVBD and to maintain oocyte-cumulus communication as assessed by transfer of a dye (Lucifer Yellow) from the oocyte to cumulus cells. Then, we assessed the effects of steroid hormones and NPPC, alone and in combination in a pre-IVM culture, on embryo production. The combination of NPPC with steroids delayed GVDB, increased natriuretic peptide receptor 2 (NPR2) mRNA abundance in cumulus cells during culture, and maintained oocyte-cumulus communication at levels not different from non-cultured controls. The addition of steroids and/or NPPC to a pre-IVM culture did not alter blastocyst rates after IVF, but supplementation with steroids increased blastocyst total cell number. The present study provides evidence, for the first time in cattle, that steroids interact with NPPC to regulate oocyte nuclear maturation and oocyte-cumulus communication, and improve oocyte developmental competence.

Effect of kit ligand on natriuretic peptide precursor C and oocyte maturation in cattle.

In vitro maturation (IVM) of oocytes in cattle is inefficient, and there is great interest in the development of approaches to improve maturation and fertilization rates. Intraovarian signalling molecules are being explored as potential additives to IVM media. One such factor is kit ligand (KITL), which stimulates the growth of oocytes. We determined if KITL enhances oocyte maturation in cattle. The two main isoforms of KITL (KITL1 and KITL2) were expressed in bovine cumulus-oocyte complexes (COC), and levels of mRNA increased during FSH-stimulated IVM. The addition of KITL to the culture medium increased the percentage of oocytes that reached meiosis II but did not affect cumulus expansion after 22 h of IVM. Addition of KITL reduced the levels of mRNA encoding natriuretic peptide precursor C (NPPC), a protein that holds oocytes in meiotic arrest, and increased the levels of mRNA encoding YBX2, an oocyte-specific factor involved in meiosis. Removal of the oocyte from the COC resulted in increased KITL mRNA levels and decreased NPPC mRNA levels in cumulus cells, and addition of denuded oocytes reversed these effects. Taken together, our results suggest that KITL enhances bovine oocyte nuclear maturation through a mechanism that involves NPPC, and that the oocyte regulates cumulus expression of KITL mRNA.

Cryotolerance and global gene-expression patterns of Bos taurus indicus and Bos taurus taurus in vitro- and in vivo-produced blastocysts.

In a 2×2 factorial experimental design, embryo development, cryotolerance and global gene expression of Nellore (Bos taurus indicus) and Simmental (Bos taurus taurus) blastocysts produced in vitro (IVP) and in vivo (multiple ovulation derived embryo, MODE) were assessed. Blastocyst production was higher in Nellore than in Simmental (47.7±2.0% vs 27.0±2.0%) cows. The total numbers of ova or embryos recovered (5.5±0.9 vs 3.7±0.8) and transferable embryos (3.8±1.0 vs 2.3±0.8) per cow were not different between breeds. Simmental and MODE (34.6% and 38.5%, n=75 and 70) blastocysts had higher survival rates after cryopreservation compared with Nellore and IVP (20.2% and 18.1%, n=89 and 94) embryos, respectively. Differences between transcriptomes were addressed by principal-component analysis, which indicated that gene expression was affected by subspecies (158 genes), origin (532 genes) and interaction between both subspecies and origin (53 genes). Several functional processes and pathways relevant to lipid metabolism and embryo viability involving differentially expressed genes were identified. The lipid metabolism-related genes were upregulated in Simmental (AUH and ELOVL6) and IVP (ACSL3 and ACSL6) blastocysts. The expression profiles of genes related to mitochondrial metabolism (ATP5B), oxidative stress (GPX4), apoptosis (DAD1, DAP, PRDX2), heat shock (HSPA5), pregnancy (IFNT2, PAG2) and cell differentiation (KRT18) varied between experimental groups.

Paternal Age Amplifies Cryopreservation-Induced Stress in Human Spermatozoa.

The global fall in male fertility is a complicated process driven by a variety of factors, including environmental exposure, lifestyle, obesity, stress, and aging. The availability of assisted reproductive technology (ART) has allowed older couples to conceive, increasing the average paternal age at first childbirth. Advanced paternal age (APA), most often considered male age ≥40, has been described to impact several aspects of male reproductive physiology. In this prospective cohort study including 200 normozoospermic patients, 105 of whom were ≤35 years (non-APA), and 95 of whom were ≥42 years (APA), we assessed the impact of paternal age on different endpoints representative of sperm quality and cryopreservation tolerance. Non-APA patients had superior fresh semen quality; DNA fragmentation was notably increased in APA as compared to non-APA individuals (21.7% vs. 15.4%). Cryopreservation further increased the DNA fragmentation index in APA (26.7%) but not in non-APA patients. Additionally, APA was associated with increased mtDNAcn in both fresh and frozen/thawed sperm, which is indicative of poorer mitochondrial quality. Cryopreservation negatively impacted acrosome integrity in both age groups, as indicated by reduced incidences of unreacted acrosome in relation to fresh counterparts in non-APA (from 71.5% to 57.7%) and APA patients (from 75% to 63%). Finally, cryopreservation significantly reduced the phosphorylation status of proteins containing tyrosine residues in sperm from young males. Therefore, the present findings shed light on the effects of paternal age and cryopreservation on sperm quality and serve as valuable new parameters to improve our understanding of the mechanisms underlying sperm developmental competence that are under threat in current ART practice.

Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus-oocyte complexes.

Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus-oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2.

Maternal protein restriction during pregnancy affects gene expression and immunolocalization of intestinal nutrient transporters in rats.

Intrauterine dietary restriction may cause changes in the functioning of offspring organs and systems later in life, an effect known as fetal programming. The present study evaluated mRNA abundance and immunolocalization of nutrient transporters as well as enterocytes proliferation in the proximal, median and distal segments of small intestine of rats born to protein-restricted dams. Pregnant rats were fed hypoproteic (6% protein) or control (17% protein) diets, and offspring rats were evaluated at 3 and 16 weeks of age. The presence of SGLT1 (sodium-glucose co-transporter 1), GLUT2 (glucose transporter 2), PEPT1 (peptide transporter 1) and the intestinal proliferation were evaluated by immunohistochemical techniques and the abundance of specific mRNA for SGLT1, GLUT2 and PEPT1 was assessed by the real-time PCR technique. Rats born to protein-restricted dams showed higher cell proliferation in all intestinal segments and higher gene expression of SGLT1 and PEPT1 in the duodenum. Moreover, in adult animals born to protein-restricted dams the immunoreactivity of SGLT1, GLUT2 and PEPT1 in the duodenum was more intense than in control rats. Taken together, the results indicate that changes in the small intestine observed in adulthood can be programmed during the gestation. In addition, they show that this response is caused by both up-regulation in transporter gene expression, a specific adaptation mechanism, and intestinal proliferation, an unspecific adaptation mechanism.