High fat diet causes distinct aberrations in the testicular proteome.

Diet has important effects on normal physiology and the potential deleterious effects of high fat diets and obesity on male reproductive health are being increasingly described. We conducted a histological review of the effects of chronic high fat (HF) diet (using a mouse model fed a 45% fat diet for 21 weeks) with a discovery proteomic study to assess for changes in the abundance of proteins in the testis. Mice on a HF diet became obese and developed glucose intolerance. Using mass spectrometry, we identify 102 proteins affected in the testis of obese mice. These included structural proteins important for the blood testis barrier (filamin A, FLNA), proteins involved in oxidative stress responses (spermatogenesis associated 20, SPATA-20) and lipid homoeostasis (sterol regulatory element-binding protein 2, SREBP2 and apolipoprotein A1, APOA1). In addition, an important regulator protein paraspeckle component 1, PSPC-1, which interacts with the androgen receptor was significantly downregulated. Proteomic data was validated using both Western blotting and immunostaining which confirmed and localised protein expression in both mouse and human testis using biopsy specimens. This study focused mainly on the abnormalities that occurred at the protein level and as a result, we have identified several candidate proteins and conducted pathway analysis around the effects of HF diet on the testis providing novel insights not previously described. Some of the identified targets could be targeted therapeutically and future work is directed in this area.

New challenges in human in vitro fertilization.

This review assesses some scientific and ethical problems with human in vitro fertilization. Improved selection of viable embryos, better culture conditions, and greater understanding of the uterine environment will increase success and prevent multiple pregnancy. Further advances will also improve oocyte cryopreservation, in vitro maturation of oocytes, knowledge of sperm function, and sperm microinjection. Preimplantation diagnosis will help avoid genetic diseases and increase understanding of embryonic defects and the viability of zygotes. The greatest ethical problem with all these developments seems to be delivery of these complex treatments when health-care resources are increasingly limited.

Culture of human preimplantation embryos to the blastocyst stage: a comparison of 3 media.

Following culture for 2 days in Earle's balanced salt solution (EBSS), human embryos which remained after transfer were cultured in one of 3 media for 4 days, from the 2- to 4-cell stage to the blastocyst stage. Sibling embryos were divided equally between treatments. Throughout the 4 day culture period, embryos were assessed for morphology and development, as well as uptake and production of energy substrates. Cell numbers in the inner cell mass and trophectoderm were determined for embryos which reached the blastocyst stage. No significant effect was observed in the extent or rate of development to the 8-cell, morula or blastocyst stage between treatments. Uptake of pyruvate was related to concentration in the medium and no differences in glucose uptake were observed between media. Endogenous energy metabolism, as measured by lactate production, was significantly higher in Ham's F12 than in EBSS from day 3.5 onwards. Blastocyst cell numbers were also increased; 79.6+/-7.7 in Ham's F12 (n=17) and 57.8+/-5.2 in EBSS (n=19), p<0.05. Of the embryos which reached the blastocyst stage by day 5, 36% (14/25) had degenerated by day 6 in EBSS compared to only 19% (5/27) in Ham's F12 (p=0.06). Slightly higher rates of embryo survival between day 5 and 6 in Ham's F12 may account for the observed increase in blastocyst cell number. The results do not suggest that improved embryo development can be obtained using human tubal fluid or Ham's F12, in preference to EBSS during early cleavage stages, but the use of Ham's F12 may improve embryo survival at later stages of development.

Insulin-like growth factor-I (IGF-I) inhibits production of IGF-binding protein-1 while stimulating estradiol secretion in granulosa cells from normal and polycystic human ovaries.

We have investigated the actions of FSH and insulin-like growth factor-I (IGF-I) on the production of IGF-binding protein-1 (IGFBP-1) by granulosa cells from unstimulated normal and polycystic (PCO) human ovaries and related these effects to those on estradiol (E2). IGFBP-1 concentrations were measured in granulosa cell-conditioned medium (48-h culture with 10(-7) M testosterone) and follicular fluid. IGF-I (50 ng/mL), in the absence of FSH, stimulated E2 production by granulosa cells from both normal and polycystic ovaries, and there was a synergistic action between IGF-I and FSH. Granulosa cells secreted IGFBP-1 in concentrations ranging from 20-500 pg/1000 cells.48 h, with cells from two of four normal and three of four polycystic ovaries showing a dose-related increase in IGFBP-1 production in response to FSH. In contrast, the addition of as little as 100 pg/mL IGF-I to cells incubated with testosterone or testosterone plus FSH, caused complete inhibition of IGFBP-1 production. FSH treatment produced the expected dose-related increase in E2 accumulation. IGFBP-1 was detectable in fluid from all sizes of follicle tested, but there was no correlation of IGFBP-1 concentrations with follicle size and no difference between normal and polycystic ovaries. These data indicate that IGFBP-1 and E2 are differentially regulated by IGF-1 in the human ovary.

Estradiol production by granulosa cells of normal and polycystic ovaries: relationship to menstrual cycle history and concentrations of gonadotropins and sex steroids in follicular fluid.

The underlying cause of anovulation in polycystic ovary syndrome is unknown. Circulating levels of immuno- and bioactive FSH are within the normal range, and the follicles contain measurable levels of bioactive FSH. The aim of this study was to compare estradiol (E2) production in response to FSH by granulosa cells from normal ovaries with those from polycystic ovaries derived from both anovulatory (anovPCO) and ovulatory subjects (ovPCO). Intrafollicular levels of immunoactive FSH, E2, and androstenedione in follicles of less than 12 mm were also measured. Follicular fluid steroid concentrations were obtained from 41 pairs of normal ovaries and 23 pairs of polycystic ovaries (8 anovPCO and 15 ovPCO). In size-matched follicles from each group there were no significant differences in follicular fluid FSH or E2 concentrations, but androstenedione levels were significantly higher in 5- to 11-mm follicles from ovPCO than in corresponding follicles from normal ovaries. Dose responses to FSH were determined in granulosa cells derived from 9 pairs of normal ovaries, 7 anovPCO, and 8 ovPCO. Cells from anovPCO produced 6- to 10-fold more E2 in response to FSH than normal cells, although there was no significant difference in the ED50 values. The response in cells from ovPCO was reduced compared to normal, but this difference did not reach significance. In summary, as judged by their FSH and E2 contents, polycystic ovaries do not have a higher proportion of atretic follicles than normal. Indeed, cells from anovPCO are hyperesponsive to FSH in vitro. This could be explained by stimulation of aromatase in vivo by either paracrine or, more probably, by endocrine factors, of which insulin is an arguable candidate.

Cellular basis of luteal steroidogenesis in the human ovary.

A primary monolayer cell culture system was developed to investigate human corpus luteum (CL) function in vitro. Steroidogenic cells were isolated by collagenase dispersal and Percoll density-gradient fractionation from CLs enucleated at progressive stages of the luteal phase (tubal surgery patients). 'Pure' granulosa-lutein cells were aspirated from ovulatory follicles at mid-cycle (in-vitro fertilization patients). The steroidogenic capacity (progesterone/20 alpha-dihydroprogesterone biosynthesis and aromatase activity) of isolated luteal cells was assessed in relation to CL development. Basal luteal cell steroidogenesis was maximal at around the expected time of ovulation and declined with CL age during the luteal phase. Conversely, human chorionic gonadotrophin (hCG)-responsive steroidogenesis was initially undetectable but developed as the luteal phase progressed. These results show that luteal cell steroidogenesis becomes increasingly dependent upon gonadotrophic support with CL age. This is evidence that functional luteolysis in human ovaries (1) is pre-programmed to occur at the cellular level, (2) is initiated automatically at the time of ovulation and (3) is reversed at the time of CL 'rescue' in early pregnancy by the direct action of trophoblastic hCG on steroidogenic luteal cells. The culture system described should be of value in further defining the control of human CL form and function at the cellular level.

Serum oestradiol and preovulatory follicular development before in-vitro fertilization.

Twenty-three in-vitro fertilization (IVF) treatment cycles (four unstimulated and 19 clomiphene-stimulated) were assessed retrospectively to discern relationships among serum oestradiol (OE2) titre on the day that human chorionic gonadotrophin (hCG) was given and the number and size of ovulatory follicles available for aspiration of oocytes during laparoscopy 32-38 h after hCG injection. Since 12 of the cycles succeeded to the stage of embryo replacement and two normal term pregnancies resulted, the series as a whole offers a useful referent data base. When only one ovulatory follicle developed (n = 8) the average volume of aspirated follicular fluid was approximately 6 ml, equivalent to a follicular diameter between 22 and 23 mm. When multiple follicles developed (mean 2.7/patient, n = 15), average fluid volume/follicle was not significantly different, averaging approximately 5.5 ml. Serum OE2 titre on the morning before hCG was injected ranged between 0.9 and 5.5 nmol/l and corresponded to the number of follicles aspirated at laparoscopy. There was a highly significant linear correlation (r = 0.85, P less than 0.001) between this OE2 value (X nmol/l) and total aspirated fluid volume (Y ml) where Y = 2.07 + 3.65 X. Thus taking 6 ml as the 'typical' fluid volume, the calibration line and its 95% confidence limits could be used to establish provisional 'ideal' pre-hCG serum OE2 titre ranges corresponding to the development of one, two or three mature ovulatory follicles. This information, combined with a knowledge of the number of presumptive preovulatory follicles present (assessed by ovarian ultrasound), can aid the timing of the hCG injection before IVF.

Microsurgery of the fallopian tube: from fantasy to reality.

PIP: The author presents 4 examples of experimental research being conducted in the techniques of microsurgery in animals. The surgically modified oviduct, capture of ova by the fimbriae, adhesion formation, and the pathophysiology of damaged tubes have been dealt with through microsurgery. Similar microsurgical techniques were applied clinically and the reported results are essentially raw data to be analyzed in the future. The operating microscope is used for all infertility surgery and it can be used over a range of magnifications. Other instruments are also mentioned, e.g., telescopic spectacles. The surgical approach is dealt with--routinely employed is the Pfannenstiel incision, a large incision. Tissues are handled as little as possible since peritoneal raw areas are an important precursor of later adhesion formation. Nonabsorbable sutures are best and nylon appears to cause the least reaction. It is helpful to leave hydrocortisone acetate in the peritoneal cavity to prevent adhesions. The success of tubal anastomosis is dependent on the method used and the patient selected. For example, of those with diathermy burns, 55% conceived. With diathermic coagulation, the pelvis remains relatively clean and the tubes adhesion-free. The authors, however, maintain that isthmic-isthmic anastomosis is best. The microscope is also used in cases of fimbrial damage; however, the results are poor. Results after repeat salpingostomy following previous surgery are even worse; of 59 patients, only 8.4% had term pregnancies. The technique is superior to those results obtained with implantation. While microsurgery is a tremendously useful technique, it would be better to develop better methods of preventive medicine. Furthermore, adequate training for microsurgery should come in the form of a fellowship at an appropriate institution.^ieng

Ovum capture and fertility following microsurgical fimbriectomy in the rabbit.

Microsurgical fimbriectomy with removal of the fimbria ovarica was performed on one side in 19 rabbits. The other tube was left intact as a control. In 10 animals the new ostium was left free, some distance from the ovary. In the remainder, the ostium was fixed to the ovarian capsule. Three animals with a fixed ostium developed mild hydrosalpinx even though the neostomy was patent. Of the remaining 16 rabbits, 15 conceived and became pregnant in both uterine horns. There was no significant difference between nidation index on the two sides. It is concluded that intact fimbriae with a fimbria ovarica are not essential for normal ovum capture. It is therefore suggested that methods for clinical salpingostomy may require re-evaluation.

The effect of treatment on experimentally produced endometrial peritoneal implants.

Endometriosis was induced surgically by implanting pieces of endometrium in the uterine mesenteries of 50 rats. Their fertility was then assessed after various treatments. Ten rats with endometriosis were treated with microsurgical excision of implants, 10 with high-frequency diathermy, 10 with danazol, and 10 with intraperitoneal indomethacin. Ten more rats had implants that were left untreated. The results were compared with those of ten rats that had sham surgery with implantation of fat in the uterine mesenteries. Reproductive performance was better in control animals with sham surgery than in animals with endometrial implants. Maximum restoration of fertility was achieved with indomethacin. Microsurgery and danazol therapy both were effective in preventing residual endometriosis, but the animals tended to be less fertile after treatment. Adhesions were most pronounced after diathermy and least pronounced after microsurgery or indomethacin therapy. In rats treated with indomethacin, persistent endometriotic cysts were invariably smaller near the site of intraperitoneal injection: this suggests a local antiprostaglandin effect.

Loupes or microscope for tubal anastomosis? An experimental study.

Standardized tubal anastomoses were performed alternatively using magnifying loupes or an operating microscope in 44 rabbits. The surgeon's personal assessment of the integrity of the anastomoses was subsequently compared with the histologic result and with pregnancies and nidation indices. After an initial learning period, the surgeon became equally satisfied with his intraoperative assessment of the anastomoses in both treatment groups. Healing was marginally better when the microscope was employed. Animals which had tubal anastomosis performed with the use of the microscope became pregnant in 81% of cases, whereas when the loupes were employed only 5% became pregnant. Measurement of the nidation index showed similar trends. It is concluded that the use of the microscope will offer superior results once the surgeon has gained limited experience with its use.

Glutamine improves human preimplantation development in vitro.

OBJECTIVE: To study the effect of glutamine on human preimplantation embryo development in vitro. DESIGN: A randomized, controlled, prospective study of 138 normally fertilized and nontransferred human embryos. On day 2 after fertilization, the embryos were allocated randomly to glucose-free medium with or without 1 mM of glutamine. SETTING: A university hospital-based IVF-ET program. PATIENT(S): Couples undergoing IVF treatment. INTERVENTION(S): Embryo culture. MAIN OUTCOME MEASURE(S): Blastocyst formation, trophectoderm and inner cell mass numbers, and pyruvate uptake and lactate production by individual embryos. RESULT(S): A higher proportion of embryos reached the morula (89% versus 68%, respectively) and blastocyst (71% versus 54%, respectively) stages when cultured with glutamine compared with embryos cultured without glutamine. Blastocyst cell numbers were not statistically significantly different for embryos grown in the presence or absence of glutamine. Pyruvate uptake and lactate production were higher in the presence of glutamine throughout development; these increases were statistically significant at the blastocyst stage. CONCLUSION(S): This study demonstrates that supplementation of the culture medium with glutamine is beneficial for human preimplantation embryo development in vitro, increasing the proportion of embryos that develop to the morula and blastocyst stages.

Primary and secondary closure of ampullary salpingotomy compared in the rabbit.

Because there is disagreement as to how linear ampullary salpingotomy should heal, primarily or secondarily, we compared the two methods with the use of rabbit oviducts in which conditions simulating ectopic pregnancy had been induced. Pregnancy rates (86% versus 75%), nidation indices (62% versus 54%), and percentage of adhesion-free tubes (50% versus 32%) were greater after primary closure, but these differences were not statistically significant. Ampullary tuboperitoneal fistulae occurred at the site of operation in 6.8% of tubes and impaired fertility (P less than 0.005).

Human embryo biopsy on the 2nd day after insemination for preimplantation diagnosis: removal of a quarter of embryo retards cleavage.

OBJECTIVE: To assess any reduction in viability and development in vitro after biopsy of a quarter of the cells of human embryos on day 2 after insemination. DESIGN: A prospective study in which normally fertilized surplus embryos of good morphology with two to eight cells approximately 48 hours after insemination were randomly allocated to a control or biopsied group, respectively. SETTING: In vitro fertilization (IVF) unit and laboratories of the Hammersmith Hospital, Institute of Obstetrics and Gynaecology, London University. PATIENTS, PARTICIPANTS: One hundred twenty-nine embryos from 28 infertile IVF patients. INTERVENTIONS: Follicular aspiration by ultrasound-guided transvaginal puncture and embryo biopsy by micromanipulative procedures. MAIN OUTCOME MEASURE(S): Pyruvate uptake and cell number at the blastocyst stage. RESULTS: Embryo biopsy did not have an adverse effect on either the proportion developing to the blastocyst stage (50% [32 of 64] and 47.7% [31 of 65] for the control and biopsied groups, respectively) or embryo viability, measured indirectly through pyruvate uptake. However, the proportion of embryos that reached the morula stage after day 4 (retarded embryos) was significantly higher (44%, 11 of 25 versus 8.7%, 2 of 23) in the biopsied group. The total number of cells (29.6 +/- 3.1 versus 62.4 +/- 4.7), numbers of inner cell mass (7.7 +/- 2.2 versus 24.5 +/- 1.4) and trophectoderm (24.0 +/- 5.2 versus 45.0 +/- 6.4) cells, and the inner cell mass:trophectoderm ratio (34.7 +/- 7.9 versus 59.5 +/- 11.7) were strikingly reduced at the blastocyst stage in the biopsied group. This reduction was greater in embryos that reached the morula stage after day 4. CONCLUSIONS: More investigation is needed to assess whether the detrimental effects observed were because of the biopsy method used in this study or to a high sensitivity of human embryos at early stages to manipulation in vitro.

Increased follicular fluid total and free cortisol levels during the luteinizing hormone surge.

OBJECTIVE: To determine the changes in follicular fluid (FF) total and free cortisol during the LH surge in naturally ovulating women. PATIENT(S): Twenty-six women having diagnostic laparoscopy during the follicular phase of normal menstrual cycles were selected. INTERVENTION(S): Blood samples were collected 1 day before, the day of, and 1 day after surgery and the results of serum E2 and LH were used to divide the cycles retrospectively into pre- and post-LH surge groups. Follicular fluid was collected during laparoscopy. MAIN OUTCOME MEASURE(S): Serum P, total and free cortisol, and FF volume, E2, P, total cortisol, and free cortisol were measured on the day of surgery. RESULT(S): Median serum and FF P levels were significantly higher in the post-LH surge group compared with the pre-LH surge group (0.54 versus 1.54 ng/mL [1.7 versus 4.85 nmol/L] and 5.03 versus 28.0 micrograms/mL [15.8 versus 88.0 mumol/L], respectively). Follicular fluid volume also increased significantly after the surge (2.5 versus 4.5 mL). Median serum total and free and percent free cortisol were higher after the surge, although not significantly. In contrast, FF total, free, and percent free levels increased dramatically between pre- and post-LH surge samples (4.41 versus 43.6 ng/mL [16.0 versus 158 nmol/L], 0.138 versus 6.68 ng/mL [0.5 versus 24.2 nmol/L], and 3.3% versus 15.0%, respectively; P < 0.05). CONCLUSION(S): An increase in total and free cortisol occurs in the follicle during the LH surge. Cortisol and its regulation by 11 beta-hydroxysteroid dehydrogenase therefore may exert a physiologic role in oocyte maturation or ovulation.

The significance of delayed suppression using buserelin acetate and recombinant follicle-stimulating hormone in a long protocol in vitro fertilization program.

OBJECTIVE: To determine whether the time taken to achieve ovarian suppression has an impact on ovarian responsiveness and the outcome of IVF-ET. DESIGN: Retrospective analysis. SETTING: An assisted reproduction unit at a university center. PATIENT(S): Patients undergoing a long protocol of IVF-ET that included buserelin acetate therapy initiated on day 2 of the cycle and recombinant FSH. INTERVENTION(S): Patients were divided into two groups according to the duration of buserelin acetate therapy required to achieve pituitary and ovarian suppression (group 1 = 2 weeks, n = 172; group 2 = > or =3 weeks, n = 337). MAIN OUTCOME MEASURE(S): Number of recombinant FSH ampules administered, duration of ovarian stimulation (days), ovarian response, and IVF outcome. RESULT(S): The patients in group 2 had lower mean E2 levels after 5 days and 9 days of stimulation than the patients in group 1. The number of recombinant FSH ampules administered and the number of days of stimulation required were higher in group 2 than in group 1. These differences were prominent in the subgroups of older patients (> or =36 years) and patients who had no evidence of polycystic ovaries on ultrasound examination. The number of oocytes retrieved and fertilized, the cancelation rate, and the pregnancy rate were similar in the two groups. CONCLUSION(S): Prolonged administration of a GnRH agonist to achieve suppression leads to a reduced ovarian response, particularly in women > or =36 years of age, but does not affect the success rate of IVF-ET.

Programming in vitro fertilization for a 5- or 3-day week.

OBJECTIVE: To schedule oocyte retrievals on either 5 or 3 days per week in in vitro fertilization (IVF) cycles. DESIGN: Human chorionic gonadotropin (hCG) administration was delayed by at least 24 hours in patients undergoing superovulation to avoid egg collections on weekends (group 1). Encouraged by the results, a further prospective study in which oocyte retrievals were programmed for only 3 days a week was undertaken (group 2). SETTING: Hammersmith Hospital, a tertiary referral unit. PATIENTS: All patients undergoing IVF treatment were included. MAIN OUTCOME MEASURES: To schedule oocyte retrievals on either 5 or 3 days per week. RESULTS: Only 4.0% of egg collections (12/303) occurred on weekends compared with 12.6% (22/175) before delaying hCG. In group 2 (n = 215), only four egg collections (2.1%) had to be performed out of schedule. Delaying administration of hCG had no detrimental effects. CONCLUSIONS: Delayed administration of hCG allows scheduling oocyte retrievals on either 5 or 3 days per week, leading to a substantial decrease in out-of-hours oocyte retrievals, reducing cycle costs, and facilitating efficiency. The method is applicable to assisted reproduction specialized units as well as district general hospitals that use gonadotropin-releasing hormone analogs in their superovulation regimens before IVF, gamete intrafallopian transfer, or intrauterine insemination.

Mechanical isolation and in vitro growth of preantral and small antral human follicles.

OBJECTIVE: To develop a procedure for isolating small human follicles and to determine their growth requirements. DESIGN: Preantral and early antral follicles were isolated manually, allocated randomly to experimental groups, and cultured for a few weeks. SETTING: Patients giving informed consent in hospitals. PATIENT(S): Women undergoing laparotomy or oophorectomy. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Follicular size, E2, histology. RESULT(S): Human FSH (at a dose of 1.5 U/mL) induced antral growth of follicles, and the addition of human LH (2.5 ng/mL) to human FSH stimulated growth and antral development. Histologic studies showed that most of the early antral follicles did not contain an oocyte and already had begun to undergo atresia before culturing. Levels of E2 increased in the incubation medium as the follicles increased in size, but those levels were significantly greater when the follicles contained oocytes. CONCLUSION(S): It is possible to grow small human follicles after they have been isolated manually. To develop successfully, they require a low concentration of human LH in addition to human FSH. The rate of atresia between the preantral and early antral stages in vivo is very high; therefore, it is worthwhile to develop techniques for isolating and culturing the follicles before the antral stages.