Overview of Anti-Müllerian hormone (AMH) and association with fertility in female cattle.

Anti-Müllerian hormone (AMH) is produced by granulosa cells of early-antral follicles found on the ovary. After production, it enters circulation and can be detected from a blood sample with an ELISA. Multiple works have found that circulating AMH is a reliable marker of the antral follicle population (AFP) of an animal as well as directly correlated to an animal's response to a superovulation protocol. Research has also found high repeatability within an animal's oestrous cycle. Further use of AMH may be valuable as a reproductive management tool, based on previous research linking productive life with circulating AMH in heifers and success to various breeding protocols by AMH concentration. The aim of this review was to summarize previous works describing basic function of AMH as well as explore recent research examining AMH as a reproductive tool and measurement of fertility in dairy animals.

Factors affecting embryo production in superovulated Bos taurus cattle.

Despite a long history of bovine superovulation research, significant commercial applications did not start until the early 1970s. For some 20 years thereafter, superovulation represented the primary tool for the production of cattle embryos. In the early 1990s, commercial invitro production (IVP) was initiated in cattle. Although ovum pick-up and IVP are now commercially practiced on a wide scale, superovulation and embryo recovery by flushing remain a widespread and very effective approach to the production of cattle embryos. This review covers both the history and the effects of multiple factors on superovulation in Bos taurus cattle. There are three general protocols for suitable pre-FSH programming of donors so that gonadotrophin-responsive follicles are available. Superovulation protocols vary widely based on the FSH source, the diluent used, the number and timing of FSH injections and the timing and utilisation of various prostaglandins, controlled internal progesterone releasing devices, gonadotrophin-releasing hormone, and other means of controlling follicular development and ovulation. The number of oocytes that can be stimulated to grow and ovulate within any given donor can be estimated by either ultrasound-guided sonography or by measuring concentrations of anti-Müllerian hormone in the blood. Animal-related factors that can influence the efficacy of superovulation include cattle breed, age, parity, genetics, lactational status and reproductive history. In addition, nutrition, stress, season, climate, weather and several semen factors are discussed.

Efficacy of a single measurement of plasma anti-Müllerian hormone concentration for ovum pick-up donor selection of Japanese Black heifers in herd breeding programs.

In this study, we evaluated the efficiency of a single measurement of plasma anti-Müllerian hormone (AMH) concentration in heifers in determining the number of oocytes recoverable by ovum pick-up (OPU), and compared AMH concentrations among sister heifers from the same parents. For this, blood samples from 50 embryo-transfer-derived female Japanese Black (JB) heifers (mean: 8.7 age in months) were collected and plasma AMH concentration was measured. At 13-15 months of age, both the number of follicles (2-9 mm) and the number of collected oocytes after OPU were counted and compared. Results indicated that the heifers with the highest AMH concentration had the highest number of follicles in their ovaries and gave the highest number of collected oocytes with OPU, thereby indicating that a single measurement of plasma AMH concentration is informative for the selection of OPU-donor heifers in herd breeding programs. The practice of performing a single AMH measurement may accelerate the intensive breeding of JB herds.

Determination of the relationship between serum anti-Müllerian hormone level and superovulatory response in Simmental cows.

The most significant focal points of the embryo transfer technology are as follows: the selection of donors, the response of the selected donor to the superovulation protocol and the obtained number of the transferable embryos. For this purpose, it is suggested that donor selection can be done by anti-Müllerian hormone (AMH) levels, and embryo production is evaluated. AMH is secreted by the granulosa cells of primordial, pre-antral and antral follicles below 4 mm in the ovary, independent of FSH. Therefore, the aim of this study was to investigate the relationship between serum AMH levels and the number of corpus luteum (CL), total embryos and transferable embryos that were shaped after a uniform superovulation protocol. For this reason, 48 Simmental cows, which were located at General Directory of Agricultural Enterprises (region, province, etc. instead of the general directorate), were used as donors for the embryo transfer. Blood samples were taken at random, regardless of the stage of animal's sexual cycle. AMH levels were measured by enzyme-linked fluorescent assay (ELFA) method of the miniVIDAS® (bioMérieux SA) using AMH Bovine Test Kit. According to the statistical analyses of the obtained data, AMH levels were positively correlated with CL and total embryos (p < .05). No significant correlations between AMH and transferable embryos were approved (p > .05). It was also determined that each 200 pg/ml increase in serum AMH level resulted in one increase in CL number. Overall, considering the positive correlation between AMH level and the obtained number of CL and total embryos after a superovulation treatment, it was concluded that measuring blood AMH level prior to any further costly implementation may be an effective method in donor selection.

Development of a programmable piggyback syringe pump and four-times-a-day injection regimen for superovulation in non-lactating Holstein cows.

The objectives of the present study were to develop a programmable piggyback syringe pump for bovine superovulation and to evaluate the effects of a four-times-a-day injection regimen using the pump. Non-lactating Holstein cows were treated with a total of 30 armour units of porcine FSH by injection four times a day with the pump (study, n = 9) or injection twice a day manually (control, n = 9) for four consecutive days from D10 of the estrous cycle. The pump-driven program successfully induced superovulation in all cows tested. The numbers of small (3- < 5 mm in diameter) and large (≥ 10 mm in diameter) follicles were greater in the study group on D11-13 and D14, respectively. There were fewer unovulated follicles detected on D21 (7 days after estrus) in the study group than in the control group (1.2 ± 0.4 and 3.2 ± 0.6, respectively).

Peri-implantation hormonal milieu: elucidating mechanisms of abnormal placentation and fetal growth.

Assisted reproductive technologies (ART) have been associated with several adverse perinatal outcomes involving placentation and fetal growth. It is critical to examine each intervention individually in order to assess its relationship to the described adverse perinatal outcomes. One intervention ubiquitously used in ART is superovulation with gonadotropins. Superovulation results in significant changes in the hormonal milieu, which persist during the peri-implantation and early placentation periods. Epidemiologic evidence suggests that the treatment-induced peri-implantation maternal environment plays a critical role in perinatal outcomes. In this study, using the mouse model, we have isolated the exposure to the peri-implantation period, and we examine the effect of superovulation on placentation and fetal growth. We report that the nonphysiologic peri-implantation maternal hormonal environment resulting from gonadotropin stimulation appears to have a direct effect on fetal growth, trophoblast differentiation, and gene expression. This appears to be mediated, at least in part, through trophoblast expansion and invasion. Although the specific molecular and cellular mechanism(s) leading to these observations remain to be elucidated, identifying this modifiable risk factor will not only allow us to improve perinatal outcomes with ART, but help us understand the pathophysiology contributing to these outcomes.

Melatonin promotes superovulation in sika deer (Cervus nippon).

In this study, the effects of melatonin (MT) on superovulation and reproductive hormones (melatonin, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and PRL) were investigated in female sika deer. Different doses (40 or 80 mg/animal) of melatonin were subcutaneously implanted into deer before the breeding season. Exogenous melatonin administration significantly elevated the serum FSH levels at the time of insemination compared with levels in control animals. During superovulation, the serum LH levels in donor sika deer reached their highest values (7.1±2.04 ng/mL) at the point of insemination, compared with the baseline levels (4.98±0.07 ng/mL) in control animals. This high level of LH was sustained until the day of embryo recovery. In contrast, the serum levels of PRL in the 80 mg of melatonin-treated group were significantly lower than those of control deer. The average number of corpora lutea in melatonin-treated deer was significantly higher than that of the control (p<0.05). The average number of embryos in the deer treated with 40 mg of melatonin was higher than that of the control; however, this increase did not reach significant difference (p>0.05), which may be related to the relatively small sample size. In addition, embryonic development in melatonin-treated groups was delayed.

Endometrial response of beef heifers on day 7 following insemination to supraphysiological concentrations of progesterone associated with superovulation.

Ovarian stimulation is a routine procedure in assisted reproduction to stimulate the growth of multiple follicles in naturally single-ovulating species including cattle and humans. The aim of this study was to analyze the changes induced in the endometrial transcriptome associated with superovulation in cattle and place these observations in the context of our previous data on changes in the endometrial transcriptome associated with elevated progesterone (P4) concentrations within the physiological range and those changes induced in the embryo due to superovulation. Mean serum P4 concentrations were significantly higher from day 4 to day 7 in superovulated compared with unstimulated control heifers (P < 0.05). Between-group analysis revealed a clear separation in the overall transcriptional profile of endometria from unstimulated control heifers (n = 5) compared with superovulated heifers (n = 5). This was reflected in the number of differentially expressed genes (DEGs) identified between the two groups with 795 up- and 440 downregulated in superovulated endometria. Ten times more genes were altered by superovulation (n = 1,234) compared with the number altered due to elevated P4 within physiological ranges by insertion of a P4-releasing intravaginal device (n = 124) with only 22 DEGs common to both models of P4 manipulation. Fewer genes were affected by superovulation in the embryo compared with the endometrium, (443 vs. 1,234 DEGs, respectively), and the manner in which genes were altered was different with 64.5% of genes up- and 35.5% of genes downregulated in the endometrium, compared with the 98.9% of DEGs upregulated in the embryo. In conclusion, superovulation induces significant changes in the transcriptome of the endometrium which are distinct from those in the embryo.

Relationship between plasma progesterone concentration and number of conceptuses and their growth in superovulated cattle.

Elevated concentrations of circulating progesterone (P4) in the immediate post-ovulation period are associated with advancement of conceptus elongation in cattle. Superovulated (SOV) cattle have not only elevated plasma P4 concentrations but also multiple embryos in the uterus because of the formation of multiple corpora lutea. We examined the relationship between plasma P4 concentration and uterine glucose level in the immediate post-ovulation period and the presence and growth of multiple conceptuses in SOV cattle. SOV cattle were artificially inseminated with frozen-thawed semen at standing estrus (day 0), and the conceptuses were recovered by nonsurgical flushing of the uterus on day 13. In the SOV cattle, there were quadratic relationships between plasma P4 concentration on days 4, 5 and 7 and conceptus length and between number of conceptuses in the uterus and conceptus length. These results suggest that conceptus growth in SOV cattle is regulated by both systemic P4 level and number of conceptuses and that there are ranges of plasma P4 concentrations and numbers of conceptuses in the uterus that are suitable for conceptus growth and development. Plasma P4 concentrations on days 5 and 7, but not the numbers of conceptuses, were quadratically correlated with uterine glucose levels on day 13 in SOV cattle. In addition, conceptus length was positively correlated with uterine glucose level in SOV cattle. Accordingly, regardless of the number of conceptuses in the uterus, the plasma P4 concentration was well correlated with the regulation of conceptus growth via changes in uterine glucose levels in SOV cattle.

Anti-Müllerian hormone profiles as a novel biomarker to diagnose granulosa-theca cell tumors in cattle.

This study was carried out to evaluate the blood profile and tissue expression of Anti-Müllerian hormone (AMH) as a biomarker for granulosa-theca cell tumors (GTCTs) in cattle. Five cases with unilateral ovarian GTCTs (GTCT group) were investigated in comparison to other groups of Japanese Black cows, which had either cystic ovarian disease (COD group, n=5), a functional corpus luteum on Days 9 to 11 of the estrous cycle (Day 0=estrus; CL group, n=13) or received superovulation treatment (SOT group, n=13). We used transrectal ultrasonography and measured plasma AMH, estradiol-17ß (E(2)), progesterone (P(4)) and testosterone (T) levels. Moreover, GTCT tissues were collected and examined by immunohistochemical staining (IHC) for AMH. In the GTCT group, ultrasound images of GTCTs were variable and not definitive. However, the AMH level in the GTCT group (n=3, 58.1 ± 66.3 ng/ml) was significantly higher than in the COD, CL and SOT groups (0.1 ± 0.1 ng/ml for GTCT vs. COD, P<0.05; 0.2 ± 0.1 and 0.3 ± 0.2 ng/ml, respectively for GTCT vs. CL and SOT, P<0.01). The other hormonal levels in the GTCT group had no significant differences compared with the COD or SOT group. Neoplastic granulosa cells labeled with AMH antibody clearly demonstrated a variety of tissue patterns in all cases by IHC. To the best of our knowledge, this is the first study to investigate the blood profile and IHC of AMH in bovine GTCTs. Our findings indicate that AMH may be a novel biomarker to diagnose GTCTs in cattle.

Relationship between different concentrations of the plasma progesterone at the time of FSH-P treatment and the superovulatory response in Holstein dairy heifers.

The aim of this study was to evaluate the influence of two different concentrations of plasma progesterone at the time of FSH-P treatment on the superovulatory response in dairy heifers. Sixteen reproductively sound Holstein heifers (13-15 months of age) were used in this study. Superovulatory treatment was commenced at mid-dioestrus (Day 10 ± 2 of the oestrous cycle) of the synchronized (using two injections of PGF2α, 11 days apart) oestrous cycles. Blood samples were collected on the day and the day after commencing FSH-P treatment and at oestrus for plasma progesterone determination. Heifers were grouped based on two levels of plasma progesterone; Group low progesterone (LP; ranging from 2 to 4.5 ng /ml; n = 7) and Group high progesterone (HP; ≥ 4.6 ng /ml; n = 8) at the beginning of FSH-P treatment (one heifer was excluded from the statistical analysis because of the abnormal progesterone level at oestrus). The superovulatory response in terms of mean numbers of palpable corpora lutea (ovulation rate) was significantly higher (p < 0.05) in group LP than group HP. Ovulation rate was negatively correlated (r = -0.51) with the progesterone concentration at the time of commencing FSH-P treatment (p < 0.05). Data suggest that varying concentrations of plasma progesterone at the time of FSH-P treatment may have a different effect on the outcome of superovulatory response in dairy heifers.

Relationship between the peripheral concentrations of estradiol-17ß (E2) and preovulatory characteristics of cumulus-oocyte complexes (COCs) during superovulation treatment in Japanese Black cows.

The relationship between the peripheral concentrations of estradiol-17ß (E(2)) and the preovulatory characteristics of cumulus oocyte complexes (COCs) during superovulation treatment was investigated in Japanese Black cows. A superovulation regimen with FSH treatment in a descending manner was commenced on day 7 (n=3) or day 10 (n=2) of the estrous cycle (day 0=estrus). Peripheral blood was collected to measure E(2) concentrations twice a day throughout the treatment. Ovariectomies were performed at 100 h after the initial FSH treatment in five cows. Every follicle more than 8 mm in diameter was isolated from the ovaries, and cumulus-oocyte complexes (COCs) were gently aspirated. The COCs were then separated into three groups based on the characteristics of the cumulus (compact, expanded and denuded) and subgrouped based on the stage of the nucleus in the oocytes (GV, GVBD). Plasma E(2) concentrations tended to increase gradually and reached the peak level at around 84 h (E(2)-84: n=3) or 96 h (E(2)-96: n=2) after the initial FSH treatment. The ratio of COCs with expanded cumulus was significantly higher in E(2)-84 than in E(2)-96 (P<0.01). However, there was no difference in the ratio of oocytes showing GVBD between E(2)-84 and E(2)-96 (P=0.73), and the characteristics of the cumulus did not affect the stage of the nucleus in the oocytes in either groups (compact, expanded and nude; P=0.61, 0.81 and 1.00). It was possible that the time until the peak plasma E(2) concentrations after the FSH treatment could become an indicator for the maturation of follicles and oocytes in preovulatory follicles during superovulation treatment in Japanese Black cows.

Systemic concentrations of endogenous and exogenous FSH in anoestrous ewes superstimulated with folltropin-V.

The synchronization of follicular waves with medroxyprogesterone acetate (MAP) and oestradiol-17beta (E(2)-17beta) prior to ovarian superstimulation in anoestrous ewes reduces the variability in superovulatory responses by an unknown mechanism. Follicle stimulating hormone (FSH) is a primary promoter of antral follicular development, but the relevance of circulating FSH concentrations to the superovulation performance in ewes has not been examined. Eighteen anoestrous Rideau Arcott ewes (May-June) were superovulated with Folltropin-V (porcine FSH), with (n = 8; treated ewes) or without (n = 10; control ewes) a single i.m. dose of 350 microg of E(2)-17beta, given on the sixth day of a 14-day treatment with MAP-releasing intravaginal sponges (60 mg). The superovulatory treatment, begun 6 days after E(2)-17beta injection, consisted of six i.m. applications of Folltropin-V given twice daily (at 08:00 and 16:00 h), followed by an i.m. injection of GnRH (50 microg). Blood samples collected every 8 h throughout the 3-day treatment, were analysed by radioimmunoassays for concentrations of ovine and porcine FSH, using species-specific standards and primary antibodies. Serum concentrations of oFSH were greater (p < 0.05) in the controls compared to treated ewes at 40, 64 and 72 h and the variability in mean oFSH concentrations was greater (p < 0.05) in control ewes at 40, 48, 64 and 72 h after the 1(st) Folltropin-V injection. There were no differences (p > 0.05) between the two groups in serum concentrations of pFSH. Significant correlations were recorded between the number of corpora lutea (CL) and oFSH concentrations at 8 h (r = 0.72, p < 0.05), 16 h (r=0.63, p < 0.05) and 64 h (r = 0.84, p < 0.01) after the 1(st) Folltropin-V injection. The total number of recovered embryos was positively correlated to oFSH concentrations at 56 h (r = 0.69, p < 0.05). We concluded that changes in endogenous FSH concentrations during ovarian superstimulation with pFSH might contribute to the variability in superovulatory responses in ewes.

Embryo yield and quality following dietary supplementation of beef heifers with n-3 polyunsaturated fatty acids (PUFA).

The objective of this study was to examine the effects of dietary n-3 polyunsaturated fatty acid (n-3 PUFA) supplementation on embryo yield and quality in heifers. Animals were individually offered barley straw and concentrate diets supplemented with either palmitic acid (C16:0; CON) or a partially rumen protected n-3 PUFA-enriched supplement. Following oestrous cycle synchronisation, superovulation was induced using FSH. Blood samples were collected for the measurement of fatty acids, metabolites, insulin and IGF-1. On day 7 post-insemination the number of ovulations was estimated and embryos recovered non-surgically and quality graded. At embryo recovery 50 ml of the uterine flushing was collected from each horn for fatty acid analysis. Grade 1 embryos were isolated, snap frozen in liquid nitrogen and stored at -80 degrees C. mRNA expression for six genes, LIF, BAX, Cx43 and E-CAD associated with embryo development, and PPAR-alpha and -delta, associated with lipid metabolism was analysed. The n-3 PUFA supplementation increased plasma n-3 PUFA concentration (P<0.05) and reduced n-6:n-3 PUFA ratio (P<0.05). Uterine concentration of the n-3 PUFA, eicosapentaenoic acid was increased (P<0.05) and the concentration of arachidonic acid decreased (P<0.05) following n-3 PUFA supplementation. While CON increased triglyceride concentrations, diet did not affect the other plasma metabolites, insulin or IGF-1 (P>0.05). Similarly, there was no effect of diet on superovulation rate, embryo recovery rate, embryo quality (P>0.05) or mRNA expression of the genes examined (P>0.05).

Ovarian responses, hormonal profiles and embryo yields in anoestrous ewes superovulated with Folltropin-V after pretreatment with medroxyprogesterone acetate-releasing vaginal sponges and a single dose of oestradiol-17beta.

In ruminants, superovulatory treatments started at the time of follicular wave emergence result in greater and less variable ovulatory responses and embryo yields compared with the treatments begun in the presence of a large growing antral follicle(s) from the previous waves. The progesterone-oestradiol treatment is routinely used for follicular wave synchronization in cattle. The main objective of this study was to characterize the ovarian responses, hormonal profiles and in vivo embryo production in anoestrous Rideau Arcott ewes (May-June), which were superovulated after pretreatment with medroxyprogesterone acetate (MAP)-releasing intravaginal sponges and a single dose of oestradiol-17beta (E(2)-17beta). Six days after insertion of MAP sponges, eight ewes were given an i.m. injection of 350 microg of E(2)-17beta (E(2)-17beta-treated ewes); 10 ewes were given an i.m. injection of vehicle (control ewes). Multiple-dose Folltropin-V treatment, followed by the bolus injection of GnRH (50 microg i.m.), began 6 days after E(2)-17beta/vehicle injection. Transrectal ovarian ultrasonography revealed that: (i) the interval between E(2)-17beta/vehicle injection and regression of all follicles > or =5 to 3 mm in diameter was shorter (p < 0.01; 2.6 +/- 0.4 vs 4.8 +/- 0.6 days respectively); and (ii) the interval between injection and emergence of the next follicular wave was longer (p < 0.05; 5.4 +/- 0.3 vs 1.2 +/- 0.4 days, respectively) in E(2)-17beta-treated than in control ewes. During the 6 days after injection, the mean FSH peak concentration and basal FSH concentration were lower (p < 0.01) in E(2)-17beta-treated ewes. The mean ovulation rate and the number of recovered embryos did not differ (p > 0.05) between the two groups of ewes. However, the number of luteinized unovulated follicles per ewe, and the variability in the number of luteal structures and overall embryo yield were less (p < 0.05) in E(2)-17beta-treated compared with control ewes. In conclusion, the MAP-E(2)-17beta pretreatment significantly reduced the variability in ovarian responses and embryo yields, without affecting the embryo production in superovulated anoestrous ewes.

Nuclear estrogen and progesterone receptors in the oviduct of heifers under natural and superovulated estrous cycles.

Oviducts from 22 crossbred heifers were examined for the presence of nuclear estrogen (ERalpha) and progesterone (PR) receptors at different phases (estrus, metaestrus and diestrus) of naturally occurring estrous cycles and estrous cycles during which superovulation was induced. Receptors were detected by immunohistochemistry in the epithelial cells, connective tissue and muscular layer of oviductal infundibulum, ampulla, ampullary/isthmic transition and isthmus. Epithelial ERalpha was found along the entire oviduct regardless of the cycle phase and of the circulating concentrations of 17beta-estradiol (E(2)) and progesterone (P(4)). Epithelial PR was found mainly at the ampullary-isthmic transition and isthmus and more intensely at the estrus phase but their amount was not correlated with P(4) concentrations. ERalpha in the connective tissue was more abundant at the infundibulum and ampulla, regardless of the phase of the estrous cycle and of E(2) and P(4) circulating concentrations. PR in the connective tissue was found mostly at the ampulla, regardless of the estrous cycle phase but no correlations were found between amount and P(4) concentrations. ERalpha staining intensity in the muscular layer was similar at all phases of the estrous cycle and at all anatomical segments of the oviducts. However, PR staining was more intense at the isthmus during the metaestrus phase and it was negatively correlated with P(4) concentrations. In general, data from the present research suggest that P(4) exerts an inhibitory role upon ERalpha and PR. Also, no differences were found between animals subjected or not to superovulation.

Serum biochemical parameters and embryo production during superovulatory treatment in dairy cattle.

The objective of this study was to determine the relationship between the number of transferable embryos (TE) and various blood chemistry parameters as a reflection of the metabolic state of cows after superovulatory treatment. Forty-nine Holstein cows were subjected to superovulatory treatment for commercial embryo production. At the time of embryo harvest, individual blood samples were taken from cows for biochemical analysis. All embryos including dead ones as well as non-fertilized oocytes were counted in uterine lavage. Feed samples collected daily for a period of two weeks before embryo harvest, were analyzed for mycotoxins: vomitoxin, zearalenone and T-2 toxin. On average, cows produced 9.45+/-5.60 embryos and oocytes of which 5.27+/-4.20 were TE, 0.37+/-0.80 were dead embryos and 3.82+/-3.78 were non-fertilized oocytes. Higher concentrations of Mg and K were associated with a higher production of TE (p=0.005 and p=0.043, respectively) and higher activity of creatinine kinase was associated with a lower production of TE (p=0.011).

Effects of breed on kinetics of ovine FSH and ovarian response in superovulated sheep.

Embryo production is a useful tool for ex situ conservation of endangered species and breeds, despite a high variability in the ovarian response to superovulatory treatments. The current study evaluated the incidence and mechanisms of genetic factors in such variability, by determining the pharmacokinetics and pharmacodynamics of a standard treatment with ovine FSH (oFSH) in two endangered Spanish sheep breeds (Rubia del Molar, R, and Negra de Colmenar, N) in comparison to Manchega ewes (M, control group). In the first experiment, pharmacokinetics of an i.m. single dose of 1.32 mg of oFSH was evaluated in seven animals of each breed. Plasma FSH concentrations reached their maximum at 4h post-administration in all the ewes, but several of the kinetic parameters (plasma FSH concentration at 4h post-administration, maximum plasma FSH concentration, C(max), and both the area under the plasma concentration-time curve extrapolated to the infinite, AUC(inf), and to the last moment of sampling, AUC(last)) were higher in the N group. In the second trial, 10 animals of each breed were superovulated using eight decreasing doses of oFSH (3 x 1.32 mg, 2 x 1.10 mg, and 3 x 0.88 mg). The R group, when compared to N and M, showed both a higher number of corpora lutea (13.7+/-0.6 versus 10.0+/-0.4 in N and 9.8+/-0.6 in M, P<0.05 for both) and embryos (7.9+/-0.8 versus 4.3+/-0.4 in N, P<0.05, and 6.7+/-0.5 in M, n.s.). Evaluation of pharmacokinetic and dynamic parameters showed that, although there was a trend for a higher hormone availability in R sheep, mean FSH plasma concentrations were similar between breeds (0.54+/-0.08 ng/ml for R, 0.45+/-0.05 ng/ml for N and 0.35+/-0.05 ng/ml for M). However, differences were found in the number of preovulatory follicles growing in response to the FSH treatment between R (24.4+/-2.2), M (18.9+/-1.5, n.s.) and N sheep (14.1+/-1.4; P<0.01). Thus, differences in embryo yields between breeds would be related to differences in the pattern of follicular growth in response to FSH treatment.

The regulation of plasma relaxin levels during human pregnancy.

The factors that determine the circulating levels of relaxin during pregnancy have been investigated by comparing the plasma levels of relaxin throughout pregnancy in women who became pregnant spontaneously (singleton, n = 240) or following superovulation (singleton and multifetal pregnancies (two to ten conceptuses), n = 83). Some of the women with multifetal pregnancies underwent selective fetal reduction to twin pregnancies. Relaxin levels were higher at 7-34 weeks of gestation in singleton pregnancies achieved following superovulation when compared with levels in spontaneously conceived singleton pregnancies (p < 0.05-0.001). In samples obtained between 10 and 12 weeks of gestation (before fetal reduction for the multifetal pregnancies), plasma relaxin levels correlated with fetal number (r = 0.526, P = 0.0001). Reduction in fetal number to a twin pregnancy did not alter relaxin levels. These data suggest that the circulating levels of relaxin throughout pregnancy are determined during the cycle of conception by gonadotrophin stimulation, and within the first 10 weeks of pregnancy by the luteotrophic stimulus from the conceptus. Furthermore, once corpus luteum synthesis of relaxin is established, then reduction in the luteotrophic stimulus does not appear to affect it.

Circulating gonadotrophin surge-attenuating factor from superovulated women suppresses in vitro gonadotrophin-releasing hormone self-priming.

A perifusion system based on ovine pituitary tissue explants was used to investigate the effects of follicular fluid (hFF) and serum from superovulated women on pituitary responsiveness to gonadotrophin-releasing hormone (GnRH). The specific aims of the study were to determine both if gonadotrophin surge-attenuating factor (GnSAF) bioactivity is present in the peripheral circulation as well as in the follicles of superovulated women and if GnSAF suppresses GnRH self-priming in vitro. Two pulses of GnRH, 1 h apart, produced marked peaks in LH secreted from control chambers, with GnRH self-priming evident in the significant difference between the first (134.4 +/- 1.7 - 232.1 +/- 24.0% of basal secretion) and second (183.9 +/- 15.8 - 313.9 +/- 14.0% of basal secretion) LH peaks. Both follicular fluid and serum pooled from two different groups of women produced marked suppression of the first (unprimed) and second (primed) LH peaks. The hFF reduced the first LH peak to 69.6 +/- 7.8 and 60.2 +/- 9.7% and the second LH peak to 57.4 +/- 6.7 and 42.6 +/- 6.5% of control LH secretion. Overall, the serum reduced the first and second LH peaks to 76.8 +/- 4.2 and 62.9 +/- 3.6% of control respectively. These results demonstrated that GnSAF bioactivity suppresses GnRH self-priming, and is present in both the peripheral circulation and hFF. The same material administered to dispersed ovine pituitary monolayers produced similar marked suppression of GnRH-induced LH secretion, with approximately 50-fold less GnSAF bioactivity in serum compared with hFF. Combined doses of oestradiol and progesterone, or hFF from large follicles containing little GnSAF, produced stimulation of GnRH-induced LH secretion and GnRH self-priming (second peaks 78.1 +/- 38.9 and 27.4 +/- 15.7% respectively higher than first peaks). Thus, in conclusion, GnSAF in hFF and serum markedly attenuated both unprimed and primed pituitary response to GnRH, virtually abolishing the GnRH self-priming effect.