Estradiol Valerate

A multicenter, open post-marketing study was conducted with a product for hormone therapy (CLIMEN^® = 2 mg estradiol valerate/2 mg estradiol valerate + 1 mg cyproterone acetate) using the MRS scale as outcome measure under routine conditions of office-based gynaecologists. The study was described in detail elsewhere [6 (link)].
In brief, 1801 gynaecologists from all parts of Germany participated in the study on a voluntary basis. 10,904 women who required hormone treatment were included. The median age was 49 years. Beside others, the MRS scale was documented before therapy and 6 months after starting the hormone treatment.
A specific problem was the transformation of an older MRS version into the advanced, relatively broad validated current version of MRS. The old version of the MRS was read by the physician and the patient answered to which extend she perceived suffering from a specific symptom, and if yes to which extend. The new scale is self-completed by the woman without interaction with the physician. The symptoms itself are the same in both scales. Nevertheless, this is a methodological limitation. We, however, are not interested in the absolute score values but relative changes after treatment compared to before. In addition, the scoring system of the old version was adjusted into the new coding system using a linear transformation. Additionally, one question of the old version was split into two questions – as recommended for the current version of the MRS (see later discussion on limitations of the study).
The statistical analyses were performed with the commercial statistical package SAS 8.2.

The animals were anesthetized with ketamine (75 mg/kg, i.p.). An incision measuring 2 cm in length was made in the subabdominal area. The abdominal muscles were opened and the intestine was retracted. The ureteric tube and the vascular base of ovaries were ligated, and the ovaries were removed. The muscle and skin incisions were closed with sutures and the animals were allowed to recover under a heat lamp. After recovery, the animals were allowed to acclimatize to the regular diet for one week. Then, they were randomly divided into four experimental groups. Two groups (n = 5 each) received 0.5 mg/kg/week estradiol valerate (Aburaihan Co., Tehran, Iran) in sesame oil via intramuscular injections for two weeks. Two groups (n = 5 each) received the sesame oil only. At the end of the two-week run-in period, a terminal experiment was performed under general anesthesia, during which groups of estradiol and vehicle-treated rats were treated with the AT₂R antagonist PD123319 or its vehicle, and renal vascular responses to Ang II were examined (see below). A fifth group (n = 8) was sham operated. These animals were not subjected to the terminal experiment, but body weight and uterine weight were determined two weeks after surgery to allow comparison with the other experimental groups.

Many studies had shown that 6-month-old female rat ovariectomized bilaterally was a good model for postmenopausal osteoporosis [32 (link),33 (link)]. We obtained a total of 72 6-month-old virgin Wistar rats with body weight of 310 ± 20.0 g from the Experimental Animal Center of Academy of Military Medical Sciences (SCXK-(Military) 2002-001, Beijing, China). The Institutional Ethics Committee of China Academy of Chinese Medical Sciences approved the experimental research on the animals. The acclimatized rats were either Sham-operated (SHAM, n = 12) or bilaterally ovariectomized (OVX, n = 60) using the dorsal approach [41 (link)]. The OVX rats were randomly divided into five groups: OVX group (OVX, n = 12); estradiol valerate treatment group (OVX + EV, n = 12); DG high-dose treatment group (OVX + DG-H, n = 12); DG medium-dose treatment group (OVX + DG-M, n = 12); DG low-dose treatment group (OVX + DG-L, n = 12). The rats in the EV group received estradiol valerate (1mg/tablet, Bayer China Ltd., Shanghai, China), which was dissolved in distilled water, to produce a concentration of 0.1 mg/kg body weight, which was administered daily by oral gavage. According to previous reports [42 (link),43 (link)], we use 24 mg/kg body weight/day, 48 mg/kg body weight/day and 96 mg/kg body weight/day as dosages of rats in DG-L, DG-M and DG-H, respectively. The doses of DG are equivalent to 1 time, 2 times and 4 times of the normal human dose of steroidal saponins in clinical prescription for coronary heart disease or myocardial ischemia (1.44 g/60 kg weight/day). The rats in the DG groups were administered DG (Sigma-Aldrich, Saint Louis, MO, USA, purity > 95%, dissolved in distilled water) at three dose daily by oral gavage (1 mL/100 g body weight). The rats in the SHAM and the OVX groups were administered the same volume of distilled water by oral gavage (1 mL/100 g body weight). The treatment started 1 week after surgery for 12 weeks. On the 15th day and the 3rd day before sacrifice, all of the rats received tetracycline (Sigma-Aldrich, Saint Louis, MO, USA) at 30 mg/kg body weight by intraperitoneal injection. The rats in all groups were fed standard rodent chow (Animal Center of the Fourth Military Medical University, Xi’an, China). The body weight of each rat was monitored weekly to assess the effect of the treatments.

The selection of FET regimen is performed based on patients’ conditions, including menstrual regularity, ovulation regularity, doctors’ preference, endometrial development, and the prevalence of endometriosis and adenomyosis. For instance, patients with regular menstrual cycles and ovulation mainly undergo NC-FET. Patients with ovulation disorders or impaired endometrium development often undergo HRT-FET, because these patients have trouble in preparing the endometrium with natural ovulation. Meanwhile, HRT-FET is also selected due to the convenience of scheduling the date of FET. Patients with endometriosis, adenomyosis or recurrent implantation failure mainly undergo combination of GnRH-a and HRT-FET.
In this study, patients in the NC-FET group underwent transvaginal ultrasound on days 8 to 10 of the menstrual cycle. Follicular growth was monitored through transvaginal ultrasound and measurement of serum luteinizing hormone (LH). When the leading follicle had reached a mean diameter of >17 mm and the serum LH level was 20 IU/L, the transvaginal ultrasound was performed every day until ovulation. The day of ovulation was confirmed by transvaginal ultrasound. Cleavage-stage embryo and blastocyst-stage embryo were thawed and transferred on 3 and 5 days after ovulation, respectively.
For patients in the HRT-FET group, endometrial preparation was initiated with oral estradiol valerate (Progynova; Bayer, Berlin, Germany) at a daily dose of 4 mg from day 5 of menstrual cycle. For patients with impaired endometrial development, a daily maximum dose of 6 mg oral estradiol valerate and 3 mg transdermal 17-β estradiol (Besins Healthcare, Paris, France) were given. The serum progesterone level was measured and the transvaginal ultrasound was performed 10-12 days after the usage of exogenous estrogen. When the endometrial thickness reached 7 mm or more and the serum progesterone level was <1.5 ng/mL, exogenous progesterone was added. The FET was scheduled for 5 days for cleavage-stage embryos and for 7 days for blastocyst-stage embryos.

In the present study, artificial endometrium preparation by the exogenous administration of estrogen and progesterone was used for the FET cycle. To prepare the endometrium, all patients self-administered oral estradiol valerate (France; DELPHARM Lille S.A.S.) 8 mg per day started at the third to fourth day of the menstrual period for 5 days, and then 12 mg per day for the remaining days of the cycle, which was adjusted according to the clinical situation during this period. Estrogen administration was continued until the endometrium reaches a thickness of 8 mm (determined by an ultrasonographic examination). And then progesterone was combined to initiate the secretory phase in preparation for FET. Two vitrified-warming embryos were selected for transfer on the fourth day of progesterone administration based on morphological grading according to the Gardner and Schoolcraft scale. Luteal support was routinely provided after FET for 14 days irrespective of pregnancy. For patients confirmed with a clinical pregnancy, estradiol valerate and progesterone were continued until 10 weeks of gestation, which was gradually reduced until detecting the fetal heart beats.

The vitrification of blastocyst was performed with a vitrification kit (Kitazato Company, Japan) according to the instruction manual. In TBT cycles, estradiol valerate (Bayer Pharmaceuticals, Germany) or Estradiol Gel (Oestrogel, Besins, Belgium) was administered on days 2–3 of menstruation. When the endometrial thickness was thicker than 8 mm and the duration of administration was at least 10 days, progesterone soft capsules (Utrogestan, Besins Healthcare, Belgium) were given vaginally for endometrium transforming, 1–2 blastocysts are thawed and transferred 5 days later.