Gonadotropins facilitate potential differentiation of human bone marrow mesenchymal stem cells into Leydig cells in vitro.

Infertility due to low testosterone levels has increased in recent years. This has impacted the social well-being of the patients. This study was undertaken to investigate the potential of gonadotropins in facilitating differentiation of human bone marrow mesenchymal stem cells (BMSCs) into Leydig cells in vitro. BMSCs were isolated, cultured, and their biological characteristics were observed. BMSCs were induced with gonadotropins in vitro and their ability to differentiate into Leydig cells was studied. The level of expression of 3-beta hydroxysteroid dehydrogenase (3ß-HSD) and secretion of testosterone were determined using flow cytometry and enzyme-linked immunosorbent assay, respectively, and the results were compared between the experimental and control groups. The cultured BMSCs showed a typical morphology of the fibroblast-like colony. The growth curve of cells formed an S-shape. After inducing the cells for 8-13 days, the cells in the experimental group increased in size and showed typical characteristics of Leydig cells, and the growth occurred in spindle or stellate shapes. Cells from the experimental group highly expressed 3ß-HSD, and there was a gradual increase in the number of Leydig cells. The control group did not express 3ß-HSD. The level of testosterone in the experimental group was higher than the control group (p < 0.05). Additionally, the cells in the experimental group secreted higher levels of testosterone with increased culture time. The expression of Leydig cell-specific markers in the experimental group was significantly higher (p < 0.05). With these findings, BMSCs can be considered a new approach for the treatment of patients with low androgen levels.

Does exogenous LH in ovarian stimulation improve assisted reproduction success? An appraisal of the literature.

A review of the scientific literature on the use of exogenous LH in assisted reproductive technology was performed by searching the MEDLINE, PubMed and Cochrane online databases. Scientific evidence was reviewed comparing recombinant FSH-only protocols to protocols supplemented with exogenous LH activity: human menopausal gonadotrophin (HMG), recombinant LH and mid-follicular human chorionic gonadotrophin (HCG). Studies were further compared based on pituitary suppression with gonadotrophin- releasing hormone (GnRH) antagonist and agonist protocols. Primary focus was given to randomized controlled trials and meta-analyses. Data from hypogonadotrophic hypogonadal patients demonstrated the importance of LH activity for success of assisted reproduction treatment. However, the majority of normogonadotrophic patients had adequate endogenous LH to successfully drive ovarian steroidogenesis and oocyte maturation. Exogenous LH supplementation was consistently associated with higher peak oestradiol concentrations. The use of HMG in long GnRH agonist cycles was associated with a 3­4% increase in live birth rate. There was insufficient evidence to make definitive conclusions on the need for exogenous LH activity in GnRH antagonist cycles or the benefit of recombinant LH and HCG protocols. Poor responders and patients 35 years of age and older may benefit from exogenous LH.

Natural cycle IVF: a question of semantics?

Recently there has been much discussion and presentation on IVF protocols using less stimulation or indeed none at all. Our experience with controlled natural cycle IVF over the last few years has convinced us that this is a powerful tool for many patients in the treatment of infertility. The protocol we employ has raised some questions as to whether it is natural cycle or stimulated cycle. We have reported a large series of cycles and seen no stimulatory effects of the medications used to control the cycle, thereby confirming our position that controlled natural cycle IVF is a valid option as an assisted reproduction treatment.

Luteinzing hormone activity in menotropins optimizes folliculogenesis and treatment in controlled ovarian stimulation.

Although the role that LH plays in folliculogenesis is still controversial, recent evidence points toward facilitatory actions of LH activity in ovulation induction. Thus, we compared the response to either highly purified FSH (75 IU FSH/ampoule; group A, 25 subjects) or human menopausal gonadotropin (75 IU FSH and 75 IU LH/ampoule; group B, 25 subjects) in normoovulatory GnRH agonist-suppressed women, candidates for intrauterine insemination. A fixed regimen of 2 daily ampoules of highly purified FSH or human menopausal gonadotropin was administered in the initial 14 days of treatment; menotropin dose adjustments were allowed thereafter. Treatment was monitored with daily blood samples for the measurement of LH, FSH, 17beta-estradiol (E(2)), progesterone, testosterone, hCG, inhibin A, and inhibin B, and transvaginal pelvic ultrasound was performed at 2-day intervals. Although preovulatory E(2) levels were similar, both the duration of treatment (16.1 +/- 0.8 vs. 12.6 +/- 0.5 days; P< 0.005) and the per cycle menotropin dose (33.6 +/- 2.4 vs. 23.6 +/- 1.1 ampoules; P < 0.005) were lower in group B. In the initial 14 treatment days the area under the curve of FSH, progesterone, testosterone, inhibin A, and inhibin B did not differ between the 2 groups, whereas LH, hCG, and E(2) areas under the curve were higher in group B. The occurrence of small follicles (<10 mm) and the inhibin B/A ratio in the late follicular phase were significantly reduced in group B. A nonsignificant trend toward a higher multiple gestation rate was present in group A (60% vs. 17%). We conclude that ovulation induction with LH activity-containing menotropins is associated with 1) shorter treatment duration, 2) lower menotropin consumption, and 3) reduced development of small ovarian follicles. These features can be exploited to develop regimens that optimize treatment outcome, lower costs, and reduce occurrence of complications such as multiple gestation and ovarian hyperstimulation.

Perivitelline space granularity: a sign of human menopausal gonadotrophin overdose in intracytoplasmic sperm injection.

The significance of the presence of coarse dark granules in the perivitelline space of oocytes has not been studied before. The study included 2288 intact oocytes [2063 in metaphase II (MII), 136 in metaphase I (MI), and 89 in germinal vesicle (GV)] retrieved in 206 intracytoplasmic sperm injection cycles stimulated by a long agonist protocol. The incidence of granules varied with oocyte maturity. It was detected in 34.3% and 4% of the MII and MI oocytes respectively, while none of the GV oocytes contained granules. The woman's age, hormonal values (oestradiol and progesterone), human chorionic gonadotrophin/oocyte retrieval interval, number of oocytes retrieved, and oocyte retrieval/injection interval were not related to the percentage of granular oocytes. Moreover, there was no correlation between the percentage of granular oocytes and the fertilization and cleavage rates, pregnancy outcome, as well as the implantation rate. Patients were divided into three groups according to the total human menopausal gonadotrophin (HMG) dose they received. There was a statistically significant difference between the three groups in the percentage of granular oocytes [17.4 +/- 5.2% versus 26.7 +/- 3.2% versus 45.4 +/- 4.2% in the low-dose (< 30 ampoules), intermediate dose (31-45 ampoules), and high-dose (> 45 ampoules) groups respectively]. We conclude that granularity in the perivitelline space is probably a physiological phenomenon related to the maturational events in oocytes and enhanced by exposure to high dosages of HMG.

Clinical applications of growth hormone for ovarian stimulation.

The realization of the existence of an intra-ovarian regulating mechanism involving insulin-like growth factor-I (IGF-I), a mediator of growth hormone (GH) action that augments the ovarian response to gonadotrophins, has prompted a number of clinical trials exploring the use of GH as an adjuvant for ovarian stimulation with human menopausal gonadotrophin. A critical review of these studies pinpoints a select group of infertile patients who may benefit from this co-treatment, particularly those who have a surgical, pathological or medically induced dysfunction of GH kinetics. The mechanism of this action, the effective dose needed and the implications regarding the interface of GH, IGF-I and ovarian physiology and pathology are now becoming clearer. A greater understanding of GH action on the ovary may in future benefit patients afflicted by anovulatory infertility and those requiring ovulation induction for in-vitro fertilization.

Ovulation induction protocols.

Ovulation induction protocols for oocyte retrieval have evolved from clomiphene citrate/human menopausal gonadotropins (menotropins) to human menopausal gonadotropins alone and, finally, to a combination of human menopausal gonadotropins and an agonist of gonadotropin-releasing hormone (GnRH-a). The almost abandonment of clomiphene use is due to findings from studies that showed reduced implantation due to the antiestrogen effect of clomiphene. The use of GnRH-a was introduced to maintain low levels of luteinizing hormone late in follicular development to prevent premature ovulation or premature senescence of the oocyte. The GnRH-a increases oocyte yield in poor responders, decreases cycle cancellations, improves the rate of pregnancy, and allows some control over the timing of retrieval. Attempts to use the GnRH-a to stimulate follicle maturation in a "short protocol" have resulted in variable and sometimes poor results. Therefore, the long GnRH-a/human menopausal gonadotropin protocol is currently used for most patients to prepare for oocyte retrieval.

Comparative in vitro and in vivo studies on the biological characteristics of recombinant human follicle-stimulating hormone.

The in vitro and in vivo activities of recombinant human FSH (recFSH) produced by a Chinese hamster ovary cell line were studied and compared with those of natural FSH preparations. The specific FSH activities of recFSH established by immunoassay and in vivo bioassay were greater than 10,000 IU/mg protein and considerably higher than the activities of tested urinary FSH references, while the in vivo bio/immuno ratios of these preparations were not significantly different. Compared to a highly purified pituitary standard (IS 83/575), recFSH had a comparable high specific in vivo bioactivity, but the specific immunoreactivity of IS 83/575 was about 2 times lower. In receptor displacement and in vitro bioassay studies recFSH provided dose-response curves parallel to those of pituitary and urinary FSH references. When equal amounts of immunoreactivity FSH were tested, recFSH and urinary and pituitary FSH displayed comparable activities in both assays. The in vitro bioactivity of recFSH could be neutralized effectively by each of three monoclonal antibodies raised against recFSH (alpha-specific), urinary FSH (beta-specific), and pituitary FSH (alpha beta-specific), respectively. Moreover, 50% inhibition of comparable responses induced by recFSH, urinary "pure" FSH, or pituitary FSH was established by the same amount of monoclonal antibody. These results support the structural and functional similarity of recFSH and natural FSH. To test whether recFSH is capable of inducing LH-specific biological responses, the in vitro induction of testosterone production in mouse Leydig cells was assessed. At least 16 IU recFSH/ml incubate were needed to increase testosterone production, indicating that the intrinsic LH bioactivity of recFSH is negligible (less than 0.025 mIU LH/IU FSH). The in vivo efficacy of recFSH was examined by treating immature female hypophysectomized rats during 4 days with recFSH only or with recFSH supplemented with hCG. RecFSH only treatment increased ovarian weight and aromatase activity in a dose-dependent manner. When recFSH dosages providing submaximal responses were supplemented with 1 IU hCG, both ovarian weight and aromatase activity were largely augmented. Neither recFSH nor urinary pure FSH, administered in a high dose was able to increase plasma estradiol levels, while ovarian weight and aromatase activity were increased to the same extent. However, when recFSH was supplemented with only 0.1 IU hCG, a 3-fold increase in median plasma estradiol levels was obtained. These findings support the two-cell two-gonadotropin theory, holding that both FSH and LH are required for estrogen biosynthesis, but also reveal that only very small amounts of LH activity are sufficient to increase estrogen secretion up to measurable plasma levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Urinary luteinizing hormone testing and prediction of ovulation in spontaneous, clomiphene citrate and human menopausal gonadotropin-stimulated cycles. A clinical evaluation.

A urinary luteinizing hormone test was utilized to predict ovulation in 99 spontaneous, 122 clomiphene citrate, and 82 human menopausal gonadotropin stimulated cycles. Tests were performed in early morning and evening specimens and follicular development was monitored by daily ultrasonography. A positive detection rate of 98, 97, and 94%, respectively, was obtained. Evidence of luteinized unruptured follicles was seen more frequently in stimulated cycles, concurring with negative test results. In 2 spontaneous, 1 clomiphene citrate and 5 hMG induced cycles two distinct LH surges were detected concomitant with a pattern of follicular atresia and subsequent new follicular development. Most ovulations occurred between 16 and 28 h after LH detection, significantly earlier in spontaneous than in clomiphene citrate stimulated cycles (p less than 0.02), whereas pre-ovulatory follicles were larger in the clomiphene citrate group (p less than 0.001). The mean duration of the follicular and luteal phases, as calculated from the LH peak, was substantially shorter in the hMG cycles than in the other two groups (p less than 0.001).

The luteal phase after in-vitro fertilization and related procedures.

To evaluate any beneficial effect of progesterone supplementation during the luteal phase of GIFT or IVF cycles stimulated by clomiphene citrate and HMG, two random prospective studies were performed. In the first study, a group of patients received a luteal phase supplement of 50 mg natural progesterone i.m. daily from the day of oocyte retrieval onwards. Initial results on 168 patients indicated that the pregnancy rate was similar in patients with or without progesterone supplements. No differences were found between the two groups in an analysis of pregnant and failed cycles. In a second study two different protocols of luteal phase supplementation after Buserelin-HMG stimulation were compared: natural progesterone in combination with oestradiol valerate (50 patients) or HCG supplements (41 patients). A 32% pregnancy rate per cycle was encountered in both groups. Endometrial biopsies, taken during the luteal phase from patients who did not undergo embryo replacement, revealed retarded endometrial development in most of the biopsies.

Some observations on the role of LH receptor binding inhibitor of ovine corpora lutea in ovarian processes.

Preliminary studies, both in vitro and in vivo were carried out to understand the physiological significance of LH receptor binding inhibitors isolated from ovine corpora lutea. Among the four active fractions tested, while UM-2R-III administered intraperitoneally suppressed both hMG and hCG-induced stimulation of uterine weights in immature mice, UM-2R-IX reduced the hCG-induced uterine weight. Fraction UM-2R-III inhibited both FSH and LH-stimulated cyclic AMP production by rat granulosa cells. Intraperitoneal injections of UM-2R-III and IX to adult cycling rats on the day of proestrus partially blocked ovulation. Similar i.p. treatment of cycling rats in 2 doses on day 1 of diestrus, prevented implantation/or early pregnancy (on 10th day of pregnancy) in 70 to 80% of rats. As the plasma levels of progesterone were low in the treated animals compared to the control, the decrease was attributed to the inadequate development of the corpus luteum. Of these fractions, UM-2R-IX was more effective at 5 to 10 times less doses compared to UM-2R-III. Further studies are needed to assess the route and dose of administering the active LHRBR components.

Ovarian failure without gonadotropin elevation in a patient with post-traumatic isolated hypogonadotropic hypogonadism.

A woman with secondary amenorrhea following head trauma showing isolated gonadotropin deficiency of hypothalamic origin did not respond to massive doses of hMG therapy (11 250 IU daily dose). Ovarian biopsy showed the absence of follicles, despite persistently low FSH levels. In this case the occurrence of premature ovarian failure was only suspected from the lack of response to hMG and diagnosed by ovarian histology.

[Cytologic and histologic studies of hormone dependence of the skin of the external female genitalia]. / Zytologische und histologische Untersuchungen zur Hormonabhängigkeit der Haut des äusseren weiblichen Genitales.

The value of vaginal cytology for functional hormonal diagnosis is generally recognized. Largely unknown was that the cornified cells obtained from the skin of the vulva likewise show morphologically demonstrable signs of hormonal effects. The cytological analysis of 429 cytological smears of the vulva obtained from 174 women during 88 cycles and the histological analysis of excisions of the vulva obtained at 65 female autopsies showed that the parakeratotic type of cornification is significantly more frequent in the premenopause than in the postmenopause. During the menstrual cycle a parakeratosis index can be identified which is parallel to the well known eosinophilic and karyopyknotic indices of cytological smears from the vagina and corresponds as well to the cyclic levels of estradiol in the serum. Exogenous administration of estrogen during the menopause results in a drastic increase of the parakeratotic index which is normally low following the menopause. These observations show that not only the uncornified squamous epithelium of the vagina but also the cornified squamous epithelium of the vulva reacts to endogenous and exogenous hormonal stimulation although in a different pattern.

[Contemporary physiology of the menstrual cycle. Part II. The reproductive hormones, regulatory mechanisms and cyclical hormonal changes]. / Kontemporêre fisiologie van die menstruele siklus. Deel II. Die voortplantingshormone, beheermeganismes en sikliese hormonale veranderinge.

Knowledge of the biochemistry of the hormones that regulate the normal menstrual cycle provides the clinician with a tool that allows physiological management of the patient. The biochemistry of the gonadotrophins and their roles in the physiology of the normal menstrual cycle are briefly reviewed. The present state of knowledge of the regulatory mechanisms is discussed and the hormonal events during the normal menstrual cycle in 50 patients are presented.