The impact in older women of ovarian FMR1 genotypes and sub-genotypes on ovarian reserve

We recently associated ovarian FMR1genotypes and sub-genotypes with distinct ovarian aging patterns. How they impact older females is, however, unknown. We, therefore, investigated 217 consecutive first in vitro fertilization (IVF) cycles in women >40 assessing oocyte yields, stratified for better (anti-Müllerian hormone, AMH >1.05 ng/mL) or poorer (AMH ≤ 1.05 ng/mL) functional reserve (FOR)). Mean age was 42.4 ± 2.0 years, mean AMH 0.76 ± 0.92 ng/mL and mean oocyte yield 5.3 ± 5.4. Overall, and in women with better FOR, FMR1 did not affect oocyte yields. With poorer FOR (AMH ≤ 1.05 ng/mL) women with het-norm/high, however, demonstrated higher oocyte yields (5.0 ± 3.8) than those with het-norm/low sub-genotype 3.1 ± 2.5; P = 0.03), confirmed after log conversion. Known associated with low FOR at young age, het-norm/high, thus, appears to preserve FOR into older age, and both het sub-genotypes appear to expand female reproductive lifespan into opposite directions.     

Association of FMR1 genotypes with in vitro fertilization (IVF) outcomes based on ethnicity/race

The FMR1 gene, mapping to an area of the X chromosome closely associated with autoimmunity also affects ovarian reserve, with specific genotypes associated with distinct ovarian aging patterns. They, therefore, could also be associated with differences of in vitro fertilization (IVF) outcomes, reported between races/ethnicities. We analyzed 339 consecutive IVF patients, 232 Caucasian, 59 African and 48 Asian, for FMR1 genotypes, and tested by multiple logistic regressions for associations between race/ethnicity, FMR1 genotype, autoimmunity and pregnancy chances with IVF. FMR1 genotypes were predictive of pregnancy (P = 0.046), het-norm/low most significantly and with decreasing chance in comparison to norm genotypes (OR 0.44; 95% CI 0.23–0.85; P = 0.014). Race/ethnicity was, overall, independently associated (P = 0.03), African demonstrating decreased odds in comparison to Caucasian (OR 0.33. 95%CI 0.13–0.79; P = 0.014). Autoimmunity did not differ but interaction of autoimmunity with FMR1 genotype almost reached significance (P = 0.07). Logistic regression with race/ethnicity and interaction between FMR1 genotype and autoimmunity in the model, demonstrated 2.5-times the odds of being associated with autoimmune positivity (OR 2.5, 1.34–4.55; P = 0.004). FMR1 genotypes offer a possible explanation for differences in IVF outcomes between races/ethnicities.     

Discordances between follicle stimulating hormone (FSH) and anti-Müllerian hormone (AMH) in female infertility

Background

Follicle stimulating hormone (FSH) and anti-Müllerian hormone (AMH) represent the two most frequently utilized laboratory tests in determining ovarian reserve (OR). This study determined the clinical significance of their concordance and discordance in female infertility patients.

Methods

We investigated 366 consecutive infertility patients (350 reached IVF), excluding women with polycystic ovarian syndrome (PCOS). They were considered to have normal FSH and AMH if values fell within age-specific (as-) 95% confidence intervals (CI), and to suffer from diminished ovarian reserve (DOR) if FSH exceeded and/or AMH fell below those. The two hormones, thus, could be concordant (Group I), both normal (IA) or abnormal (IB), show normal AMH/abnormal FSH (Group II) or normal FSH/abnormal AMH (Group III). Oocyte yields, stratified for age categories, were then studied in each group as reflection of OR.

Results

Oocyte yields significantly decreased from groups IA to II to III and IB. Predictive values of as-FSH/AMH patterns changed, however, at different ages. Except at very young and very old ages, normal as-AMH better predicted higher oocytes yields than normal as-FSH, though above age 42 years normal as-FSH predicts good oocyte yields even with abnormally low AMH. Under age 42 discrepancies between as- FSH and as-AMH remain similarly predictive of oocyte yields at all ages.

Discussion

Concordances and discordances between as-FSH and as-AMH improve OR assessments and predictability of oocyte yields in IVF.

     

Defining ovarian reserve to better understand ovarian aging

Though a widely utilized term and clinical concept, ovarian reserve (OR) has been only inadequately defined. Based on Medline and PubMed searches we here define OR in its various components, review genetic control of OR, with special emphasis on the FMR1 gene, and discuss whether diminished OR (DOR) is treatable. What is generally referred to as OR reflects only a small portion of total OR (TOR), a pool of growing (recruited) follicles (GFs) at different stages of maturation. Functional OR (FOR) depends on size of the follicle pool at menarche and the follicle recruitment rate. Both vary between individuals and, at least partially, are under genetic control. The FMR1 gene plays a role in defining FOR at all ages. Infertility treatments have in the past almost exclusively only centered on the last two weeks of folliculogenesis, the gonadotropin-sensitive phase. Expansions of treatments into earlier stages of maturation will offer opportunity to significantly improve ovarian stimulation protocols, especially in women with DOR. Dehydroepiandrosterone (DHEA) may represent a first such intervention. Data generated in DHEA-supplemented women, indeed, suggest a new ovarian aging concept, based on aging of ovarian environments and not, as currently is believed, aging oocytes.     

Can egg donor selection be improved? - a pilot study

Background

Accurate assessments of ovarian reserve (OR) in egg donor candidates are crucial for maximal donor selection. This study assesses whether recently reported new methods of OR assessment by age-specific (as-), rather than non-as (nas-) hormones, follicle stimulating hormone (FSH) and anti-Müllerian hormone (AMH), and triple nucleotide (CGG) repeats on the FMR1 (fragile X) gene have the potential of improving egg donor selection.

Methods

Seventy-three consecutive egg donor candidates (candidates), amongst those 21 who reached egg retrieval (donors), were prospectively investigated for as-FSH, as-AMH and number of CGG repeats. Abnormal findings were assessed in candidates and donors and oocyte yields in the latter were statistically associated with abnormal FSH and AMH (>/< 95% CI of as-levels) and with normal/abnormal numbers of CGG repeats (normal range 26-32).

Results

Amongst candidates mean as-AMH was 3.8 +/- 2.8 ng/mL (37.0% normal, 3.0 +/- 0.7 ng/mL; 26.6% low, 1.5 +/- 0.5 ng/mL; and 37.0% high, 5.8 +/- 2.2 ng/mL). AMH among donors was 4.2 +/- 1.7 ng/mL (33.3% normal, 14.3% low, and 52.4% high), yielding 17.8 +/- 7.2 oocytes, 42.9% in normal range (10-15), 9.5% in low (less than or equal to 9) and 47.6.% in high range (16-32). Candidates in 41.9% and donors in 38.1% demonstrated normal CGG counts; the remained were mostly heterozygous abnormal.

Discussion

Prospective assessment of even carefully prescreened candidates and donors still demonstrates shortcomings on both ends of the OR spectrum. Utilization of ovarian reserve testing methods, like as-hormones and CGG repeats on the FMR1 gene have potential of improving candidate selections.

     

FMR1 genotype with autoimmunity-associated polycystic ovary-like phenotype and decreased pregnancy chance

The FMR1 gene partially appears to control ovarian reserve, with a specific ovarian sub-genotype statistically associated with a polycystic ovary (PCO)- like phenotype. Some forms of PCO have been associated with autoimmunity. We, therefore, investigated in multiple regression analyses associations of ovary-specific FMR1 genotypes with autoimmunity and pregnancy chances (with in vitro fertilization, IVF) in 339 consecutive infertile women (455 IVF cycles), 75 with PCO-like phenotype, adjusted for age, race/ethnicity, medication dosage and number of oocytes retrieved. Patients included 183 (54.0%) with normal (norm) and 156 (46%) with heterozygous (het) FMR1 genotypes; 133 (39.2%) demonstrated laboratory evidence of autoimmunity: 51.1% of het-norm/low, 38.3% of norm and 24.2% het-norm/high genotype and sub-genotypes demonstrated autoimmunity (p=0.003). Prevalence of autoimmunity increased further in PCO-like phenotype patients with het-norm/low genotype (83.3%), remained unchanged with norm (34.0%) and decreased in het-norm/high women (10.0%; P<0.0001). Pregnancy rates were significantly higher with norm (38.6%) than het-norm/low (22.2%, p=0.001). FMR1 sub-genotype het-norm/low is strongly associated with autoimmunity and decreased pregnancy chances in IVF, reaffirming the importance of the distal long arm of the X chromosome (FMR1 maps at Xq27.3) for autoimmunity, ovarian function and, likely, pregnancy chance with IVF.     

BRCA1/2 Mutations Appear Embryo-Lethal Unless Rescued by Low (CGG n<26) FMR1 Sub-Genotypes: Explanation for the “BRCA Paradox”?

BRCA1/2 mutations and recently described constitutional FMR1 genotypes have, independently, been associated with prematurely diminished ovarian reserve. Whether they interrelate in distribution, and whether observed effects of BRCA1/2 and FMR1 on ovaries are independent of each other, is unknown. In a prospective comparative cohort study, we, therefore, investigated the distribution of constitutional FMR1 genotypes, normal (norm), heterozygous (het) and homozygous (hom), and of their respective sub-genotypes (high/low), in 99 BRCA1/2 mutation-positive women and 410 female controls to determine whether distribution patterns differed between study and control patients. In contrast to controls, BRCA1/2 carriers demonstrated almost complete absence of all constitutional FMR1 genotypes except for sub-genotypes with low (CGG _n<26) alleles. Cross tabulation between BRCA1/2-positive patients and controls confirmed significant group membership, related to FMR1 distribution (P<0.0001). These results offer as most likely explanation the conclusion that BRCA1/2 mutations are embryo-lethal, unless rescued by low (CGG _n<26) FMR1 sub-genotypes, present in approximately one quarter of all women. Women with low FMR1 sub-genotypes, therefore, should reflect increased BRCA1/2-associated cancer risks, while the remaining approximately 75 percent should face almost no such risks. If confirmed, this observation offers opportunities for more efficient and less costly BRCA1/2 cancer screening. The study also suggests that previously reported risk towards prematurely diminished ovarian reserve in association with BRCA mutations is FMR1-mediated, and offers a possible explanation for the so-called “BRCA paradox” by raising the possibility that the widely perceived BRCA1/2-associated tumor risk is actually FMR1-mediated.     

Follicle stimulating hormone and anti-Müllerian hormone per oocyte in predicting in vitro fertilization pregnancy in high responders: a cohort study

Background

Follicle stimulating hormone (FSH) and Anti-Müllerian hormone (AMH) are utilized to differentiate between good and poor response to controlled ovarian hyperstimulation. Their respective roles in defining functional ovarian reserve remain, however, to be elucidated. To better understand those we investigated AMH and FSH per oocyte retrieved (AMHo and FSHo).

Methodology/Principal Findings

Three-hundred and ninety-six women, undergoing first in vitro fertilization cycles, were retrospectively evaluated. Women with oocyte yields >75^th percentile for their age group were identified as high responders. In a series of logistic regression analyses, AMHo and FSHo levels were then evaluated as predictive factors for pregnancy potential in high responders. Patients presented with a mean age of 38.0±5.0 years, mean baseline FSH of 11.8±8.7 mIU/mL and mean AMH of 1.6±2.1 ng/mL. Those 88 women, who qualified as high responders, showed mean FSH of 9.7±6.5 mIU/mL, AMH of 3.1±3.1 ng/mL and oocyte yields of 15.8±7.1. Baseline FSH and AMH did not predict pregnancy in high responders. However, a statistically significant association between FSHo and pregnancy was observed in high responders, both after univariate regression (p = 0.02) and when adjusted for age, percentage of usable embryos, and number of embryos transferred (p = 0.03). Rate of useable embryos also significantly affected pregnancy outcome independently of FSHo (p = 0.01). AMHo was also associated with clinical pregnancy chances in high responders (p = 0.03) and remained significant when adjusted for usable embryos and number of embryos transferred (p = 0.04).

Conclusions

AMHo and FSHo are predictive of pregnancy potential in high responders, but likely reflect different responsibilities in recruitment and maturation of growing follicle cohorts.     

Bovine model of reproductive aging: response to ovarian synchronization and superstimulation

The responsiveness of the hypothalamo-pituitary axis to steroid treatments for ovarian synchronization and the ovarian superstimulatory response to exogenous FSH was compared in 13-14 year old cows and their 1-4 year old young daughters. We tested the hypotheses that aging in cattle is associated with: (1) decreased follicular wave synchrony after estradiol and progesterone treatment; (2) delayed LH surge and ovulation in response to exogenous preovulatory estradiol treatment; (3) reduced superstimulatory response to exogenous FSH. Higher plasma FSH concentrations (P<0.01), and a tendency (P=0.07) for fewer 4-5 mm follicles at wave emergence were observed in old cows (n=10) than in young cows (n=9). The suppressive effect of estradiol/progesterone treatment on FSH was similar between old and young cows. Although the preovulatory LH surge in response to estradiol treatment was delayed in old than young cows (P=0.01), detected ovulation times were not different. No difference in ovarian superstimulatory response was detected between age groups, but old cows (n=8) tended (P=0.10) to have fewer large follicles (>or=9 mm) 12 h after last FSH treatment than in young cows (n=7). We concluded that pituitary and ovarian responsiveness to estradiol/progesterone synchronization treatment was similar between old and young cows, but aging was associated with a delayed preovulatory LH surge subsequent to estradiol treatment. Old cows tended to have fewer large follicles after superstimulatory treatment than young cows.     

Anti-Müllerian hormone concentration in sheep and its dependence of age and independence of BMP15 genotype: An endocrine predictor to select the best donors for embryo biotechnologies

Embryo biotechnologies contribute significantly to the genetic enhancement of livestock, although their efficiency remains limited in sheep, mainly owing to variable ovarian responses to gonadotropins. At present, anti-Müllerian hormone (AMH), which is produced by the granulosa cells of the small antral follicles, is a reliable endocrine marker of the ovarian follicle reserve in many species. The expression of AMH in granulosa cells was shown to be stimulated by bone morphogenetic proteins (BMPs) in vitro, so a mutation affecting the BMP15 gene might modulate AMH production in vivo. The present study aimed to assess plasma AMH concentrations before puberty in two groups of Rasa Aragonesa ewes that were carrying (R+) or not carrying (++) the prolific FecX^R allele and to relate them with their AMH concentrations at adulthood. Additionally, we sought to establish in both genotypes whether AMH measurements during a laparoscopic ovum pick-up (LOPU) program could be predictive of the number of ovarian follicles (≥3 mm) and recovered cumulus-oocyte complexes (COCs). No differences in AMH were found between the R+ and ++ ewes before puberty or during the adult age. Before puberty, the AMH concentration tended to increase from 3 to 4.5 months and to decline at 6 months to levels similar to those observed later in adults (333.8 ± 73.3, 483.2 ± 135.5, and 184.1 ± 38.2 pg/mL, respectively; P < 0.1), showing a large variability between individuals and between ages. A relationship between the AMH concentrations before puberty and during adulthood was not found, likely reflecting different follicular growth dynamics. In adults, the AMH concentration at the beginning of the FSH treatment was strongly correlated with the number of punctured follicles at LOPU in R+ and ++ ewes (r = 0.75 and 0.78, respectively; P < 0.001), and it was possible to accurate determine AMH cutoff values for both genotypes to identify high-responding ewes. On average, 5.1 extra follicles and 2.7 extra COCs were expected per each 100 pg/mL increase in AMH (P < 0.0001 and P < 0.01, respectively). The repeatability of AMH concentration from session to session was 0.70 (P < 0.0001). Our results demonstrated that, regardless of age, the presence of the FecX^R allele did not affect plasma AMH levels. During adulthood, AMH proved to be a good predictor of the ovarian response to FSH stimulation. Such an indicator could therefore be used to improve the performance of embryo biotechnologies in sheep.     

Orthotopic ovarian transplantations in young and aged C57BL/6J mice

Orthotopic ovarian transplantations were done between young (6-wk-old) and aged (17-mo-old) C57BL/6J mice. The percentages of mice mating following surgery from the four possible ovarian transfer combinations were as follows: young into young, 83%; young into aged, 46%; aged into young, 83%; and aged into aged, 36%. The percentages of these mice that were pregnant 10 days following the presence of a vaginal sperm plug were as follows: young into young, 58%; young into aged, 9%; aged into young, 50%; and aged into aged, 0%. Some of the fetuses derived from matings of the above mice were dissociated and their cells prepared for chromosomal smears. No evidence of aneuploidy or mosaicism was found in fetuses derived from ovaries of young or aged mice. Aged ovaries, transferred to either young or aged recipients, were found to have fewer developing follicles and lower weight, which was most apparent in recipients that failed to mate or to get pregnant. Concentrations of luteinizing hormone, follicle-stimulating hormone (FSH), and prolactin in plasma from each of the pregnant recipients were analyzed by radioimmunoassay. The only statistical differences found between the transfer groups occurred in FSH concentrations. Plasma FSH was markedly elevated (P less than 0.005) in young recipients with ovaries transplanted from aged donors, in comparison to young recipients with ovaries from young donors. These data indicate that the aging ovary and uterus play a secondary role in reproductive failure and that the aging hypothalamic-hypophyseal complex is primarily responsible for the loss of fecundity in older female C57BL/6J mice.     

Embryo production using defined oocyte maturation and zygote culture media following repeated ovum pick-up (OPU) from FSH-stimulated Simmental heifers

To determine whether differences in ovarian follicle populations and endocrine status at ovum pick-up (OPU) influenced the quality and developmental competence of oocyte-cumulus complexes (OCC's) collected from follicle stimulating hormone (FSH)-stimulated donors, 24 Simmental heifers had their ovarian follicles aspirated via transvaginal ultrasound-guided OPU at both 15 (OPU1) and 21 (OPU2) days following a synchronised oestrus, on four consecutive occasions at 15-week intervals. More OCC's were collected during OPU1 than OPU2 (means +/- S.E.M. = 7.2 +/- 0.47 versus 5.7 +/- 0.44; P = 0.01), but the respective percentages that were of good quality (categories 1 and 2) did not differ significantly (55 +/- 3% versus 47 +/- 3%). The incidence of zygote cleavage following OCC maturation (Medium 199; protein-free), in vitro fertilization (mTALP; including 0.6% (w/v) albumin) and culture (modified SOF; protein-free) was not significantly different (mean +/- S.E.M. = 81 +/- 2% and 71 +/- 7% for OPU1 and OPU2, respectively). Corresponding blastocyst yields from good quality OCC's (24 +/- 3% and 26 +/- 4%) also did not differ. Although the same 3-day FSH regimen was used immediately prior to each OPU session, plasma FSH concentrations were consistently lower at OPU1 than OPU2 (1.3 +/- 0.28 ng/ml versus 2.5 +/- 0.45 ng/ml; P < 0.05). In contrast, plasma progesterone concentrations were higher at OPU1 (6.6 +/- 0.48 ng/ml versus 3.9 +/- 0.53 ng/ml; P < 0.001), with concentrations at OPU2 being consistent with the presence of luteal tissues, including both persistent corpora lutea and luteinised follicle remnants following OPU1. Failure of the significant differences in follicular and endocrine status between OPU1 and OPU2 to alter the developmental competence of OCC's suggests that, probably as a result of its stabilising influence on nutritionally-sensitive intraovarian regulators of oocyte competence, the constant feeding regimen had a more profound effect on oocyte quality than observed shifts in the peripheral concentrations of some reproductive hormones. Finally, the study demonstrates that it is possible to generate acceptable numbers of in vitro blastocyst-stage embryos from high genetic merit heifers using strategies which restrict reliance on protein to the in vitro fertilization stage of the production process.     

Prediction of ovarian response to exogenous gonadotropin stimulation: utilization for collection of primate oocytes for fertilization in vitro

The predictive value of 2 tests for ovarian response to controlled ovarian hyperstimulation in the cynomolgus monkey model was evaluated. The tests utilized were: 1) the cycle Day 3 (Day 1 = onset of menses) FSH value and 2) the acute estradiol (E(2)) response to a GnRH agonists (GnRHa) administered on Day 3. Both tests were performed during the cycle preceding control ovarian hyperstimulation. Subsequently, monkeys (n = 26) were stimulated with Metrodin(T) (Days 2-6, 25IU/d) and Pergonal(T) (Day 7 to hCG administration, 25IU/d). Laparoscopic oocyte retrieval was performed 32 to 34 after hCG administration. Analysis of the data revealed that Day 3 FSH values could not predict whether an animal would respond well to control ovarian hyperstimulation in a subsequent cycle (P = 0.77). However, the E(2) change 24 h post-GnRHa administration was significantly greater for animals responding well to control ovarian hyperstimulation compared with the animals deleted after 6 d of stimulation (P = 0.042). The mean change in E(2) levels in animals taken to aspiration was 97.8 pg/ml compared with only 21.6 pg/ml for the deleted animals. This differential response of E(2) production after GnRHa treatment was used to correctly identify (by discriminant analysis) 78% of the animals subsequently deleted for poor response. Thus, the increase in serum E(2) level after GnRHa, but not the basal FSH level, was found to be predictive of ovarian response to stimulation in the cynomolgus monkey.     

Study of FMR1 gene association with ovarian dysfunction in a sample from the Basque Country

Premature ovarian failure (POF) is defined as cessation of menses before the age of 40. The most significant single gene associated with POF is the Fragile X Mental Retardation 1 gene (FMR1). In the present work we screened women with fertility problems from the Basque Country in order to determine, whether in these women, FMR1 CGG repeat size in the intermediate and premutation range was associated with their pathology, and whether intermediate and premutation carriers had endocrine signs of diminished ovarian function, using the most established measure of ovarian reserve, the gonadotropin FSH. A patient sample of 41 women with ovarian insufficiency and a control sample of 32 women with no fertility problems from the Basque Country were examined. The patient sample was classified into three categories according to the results of the retrospective assessment of their ovarian function. In group 2 of patients, women with irregular cycles, reduced fecundity and FSH levels ≥ 10 IU/l, there is a significant increase in the number of intermediate and premutation FMR1 alleles (35–54 CGG repeats). In group 3 of patients, women with amenorrhea for at least four consecutive months and FSH levels ≥ 10 IU/l, a significant increase in the number of intermediate FMR1 alleles (35–54 CGG repeats) was found in patients compared with controls. In this group all the patients had a serum concentration > 40 IU/l. The results suggest that in the analysed Basque sample the FMR1 gene has a role in the aetiology of POF. However, elevated FSH levels are more related to the menstrual cycle pattern than to the CGG repeat size.     

Aromatase inhibitors in post-menopausal endometriosis

Background

Serum anti-Mullerian hormone (AMH) is currently considered the best marker of ovarian reserve and of ovarian responsiveness to gonadotropins in in-vitro fertilization (IVF). AMH assay, however, is not available in all IVF Units and is quite expensive, a reason that limits its use in developing countries. The aim of this study is to assess whether the "ovarian sensitivity index" precisely reflects AMH so that this index may be used as a surrogate for AMH in prediction of ovarian response during an IVF cycle.

Methods

AMH serum levels were measured in 61 patients undergoing IVF with a "long" stimulation protocol including the GnRH agonist buserelin and recombinant follicle-stimulating hormone (rFSH). Patients were divided into four subgroups according to the percentile of serum AMH and their ovarian stimulation was prospectively followed. Ovarian sensitivity index (OSI) was calculated dividing the total administered FSH dose by the number of retrieved oocytes.

Results

AMH and OSI show a highly significant negative correlation (r = -0.67; p = 0.0001) that is stronger than the one between AMH and the total number of retrieved oocytes and than the one between AMH and the total FSH dose.

Conclusions

OSI reflects quite satisfactory the AMH level and may be proposed as a surrogate of AMH assay in predicting ovarian responsiveness to FSH in IVF. Being very easy to calculate and costless, its use could be proposed where AMH measurement is not available or in developing countries where limiting costs is of primary importance.     

A bovine-specific FSH enzyme immunoassay and its application to study the role of FSH in ovarian follicle development during the postnatal period

The primary aim of this study was to develop a FSH enzyme immunoassay (EIA) for the bovine species. The newly developed EIA was validated for FSH determination in bovine plasma by comparison with an existing bovine FSH radioimmunoassay. The EIA detected bovine FSH with a high sensitivity (0.1 ng/ml). Cross-reactivity of the EIA was 0.01% with bovine LH, 51% with ovine FSH, <0.1% with porcine FSH and <0.01% with equine FSH. Using this EIA on different time series of plasma in cows, we have confirmed the presence of a FSH pre-ovulatory peak at estrus, of periodic FSH fluctuations accompanying the waves of terminal follicular development, and of FSH pulses, mainly asynchronous with LH ones, in the peri-ovulatory phase of the cycle. In a second objective, the EIA was used to assess the role of FSH in regulating the development of ovarian follicles up to the small antral stage in young calves. To answer this question, six calves were submitted to weekly blood sampling during their first 3 months of life, and FSH changes were studied concomitantly to those of anti-Müllerian hormone (AMH), a well-established endocrine marker of the ovarian population of small antral follicles in cows. In the ovaries of 3-month calves, the population of 3 to 5 mm follicles contained the highest intra-follicular AMH amounts, and the number of 3 to 5 mm follicles on ovaries was closely correlated with AMH concentrations in the plasma of calves at this age (r_s = 0.94). Before 3 months of age, only two out of six calves showed a clear postnatal FSH peak in plasma, and no correlation was found between plasma FSH and AMH concentrations. These results indicate that female calves undergo different patterns of FSH secretion and that postnatal activation of follicular growth up to the small antral stage appears independent and not directly related to circulating FSH levels.     

DNA polymorphic patterns and haplotype arrangements of the apo A-1, apo C-III,apo A-IV gene cluster in different ethnic groups

Summary The allelic frequency of five different restriction fragment length polymorphisms (RFLPs) in the A-1, C-III, A-IV gene region has been determined in Caucasians, Negroes, Indian Asians, and Japanese. The polymorphic sites are with Taq-1 at the 5 end of the A-1 gene, with Msp-1 in the third intron of the A-1 gene, with Pst-1 in the intergenic sequence between the A-1 and C-III genes, with Sst-1 in the 3 non-coding region of the C-III gene, and with Pvu-II in the third intron of the C-III gene. The alleles identified by three of the RFLPs showed large differences in frequency amongst the races, especially between Caucasians and non-Caucasians. Alleles of the Msp-1 polymorphism and Sst-1 polymorphism, which were rare in Caucasians (frequencies 0.03 and 0.01), were more common in Japanese (frequencies 0.37 and 0.35), Indian Asians (frequencies 0.37 and 0.26), and Negroes (frequencies 0.31 and 0.31). In contrast with a Pvu-II polymorphism one allele was rare in Japanese and in Indian Asians (frequency 0.01) but more common in Caucasians (frequency 0.11). Linkage disequilibrium was evident between some of the alleles and a total of seven haplotypes were identified among the different races.     

Effects of eCG and FSH on ovarian response, recovery rate and number and quality of oocytes obtained by ovum pick-up in Holstein cows

The goal of the present study was to compare the ovarian response, oocyte yields per animal, and the morphological quality of oocytes collected by ultrasound guided follicular aspiration from Holstein cows treated either with FSH or eCG. Twenty four normal cyclic, German Holstein cows were randomly divided into two groups. Fourteen cows received 3000 IU eCG on day-4 prior to ovum pick-up (OPU) (day 0), 2 days later (day-2), 625 microg cloprostenol was administered. On day-1 GnRH was administered i.m. and 24h later OPU (day 0) was performed. In ten cows a total dose of 500 IU follicle stimulating hormone (Pluset) was administered intramuscularly in a constant dosage for 4 days with intervals of 12h, starting on day-5. Luteolysis was induced by application of 625 microg cloprostenol on day-2. On day-1 (24h after the last FSH treatment) GnRH was administered i.m. and 24h later OPU (day 0) was performed. Ovarian follicles were visualized on the ultrasound monitor, counted and recorded. All visible antral follicles were punctured. Recovered oocytes were graded morphologically based on the cumulus investment. Average follicle number in ovaries was higher in FSH group than eCG group (p<0.05). Oocyte yields per animal did not differ between FSH and eCG groups. The proportion of grade A oocytes was higher in the FSH group in the than eCG group (p<0.05). Likewise, rate of grade C oocytes in FSH group were lower than eCG group (p<0.05). In conclusion, these results suggest that ovarian response, follicle number in ovaries and oocyte quality are affected by the type of gonadotropin and FSH is better alternative than eCG for OPU treatment.