Female Fertility Study Research Articles

Reversible effects on female rat fertility with abrocitinib, a Janus kinase 1 inhibitor.

Abrocitinib is a Janus kinase (JAK) 1 selective inhibitor approved for the treatment of atopic dermatitis. Female reproductive tissues were unaffected in general toxicity studies, but an initial female rat fertility study resulted in adverse effects at all doses evaluated. A second rat fertility study was conducted to evaluate lower doses and potential for recovery. This second study had 4 groups of 20 females each administered abrocitinib (0, 3, 10, or 70 mg/kg/day) 2 weeks prior to cohabitation through gestation day (GD) 7. In addition, 2 groups of 20 rats (0 or 70 mg/kg/day) were dosed for 3 weeks followed by a 4-week recovery period before mating. All mated females were evaluated on GD 14. No effects were observed at ≤10 mg/kg/day. At 70 mg/kg/day (29x human exposure), decreased pregnancy rate, implantation sites, and viable embryos were observed. All these effects reversed 4 weeks after the last dose. Based on these data and literature on the potential role of JAK signaling in implantation, we hypothesize that these effects may be related to JAK1 inhibition and, generally, that peri-implantation effects such as these, in the absence of cycling or microscopic changes in nonpregnant female reproductive tissues, are anticipated to be reversible.

P-161 Impact of physical activity (PA) on oocyte quality: a prospective study among IVF/ICSI patients

Abstract Study question To determine if various levels of PA have an influence on oocyte quality, ovarian response to controlled stimulation and IVF/ICSI success rate. Summary answer Globally, PA has no influence on ovarian response in IVF cycles. However, in certain subgroups (endometriosis, disovulation, normal-BMI) high PA is associated with higher response. What is known already Lifestyle is considered a key factor concerning health. According to WHO, moderate PA is an effective way to lower the risk of many pathologic conditions, although the impact of PA on female fertility is still unknown. There is scientific evidence that vigorous exercise can be detrimental for female fertility Studies evaluating the effect of PA on oocyte quality are lacking. However, murine models show that PA may have a positive impact on it. Study design, size, duration Prospective observational study. PA was evaluated before the IVF cycle, using the International PA Questionnaire (IPAQ) (n = 617), assessing frequency and duration of PA in the previous week, and using accelerometer (n = 102). Mean age of women was 35. Patients were classified into three levels of PA: low, moderate, high. The following outcome measures were analysed: number of collected oocytes, mature oocytes, ovarian response, biochemical pregnancy rate. Size: 617 infertile women undergoing IVF/ICSI cycles January 2019 - October 2020 Participants/materials, setting, methods 617 participants voluntarily fulfilled the IPAQ prior to IVF/ICSI cycle. Ethical approval was obtained from the Clinical Research Ethics Committee (CEIC E19/06). IPAQ’s results were measured in metabolic equivalent of task (METs-min/week) (Walking = 3.3 METs; Moderate PA = 4 METs; Vigorous PA = 8 METs). 1 MET = resting metabolic rate. In 102 participants PA was objectively measured for 7 consecutive days, prior to IVF cycle, using triaxial accelerometry. Setting: Human Reproduction Unit of a Universitary Hospital (Spain) Main results and the role of chance The number of collected oocytes was similar in all three groups according IPAQ (9.23 ± 7.72; 8.35 ± 5.57; 8.82 ± 6.38). Something similar happened with the number of mature oocytes (MII) (6.97 ± 5.99; 6.84 ± 4.85; 7.05 ± 5.61) and fertilized oocytes (3.72 ± 3.84; 4.16 ± 3.35; 3.98 ± 3.88). Biochemical pregnancy rate was slightly superior in high PA vs moderate vs low (38% vs 34.8% vs 29%), but without statistical significance. In the subgroup of patients having endometriosis the number of MII oocytes was significantly superior in high and moderate vs low PA (p = 0.024). In the group of disovulating women there were also more MII oocytes in high vs moderate vs low PA (p = 0.038). When performing the analysis according to accelerometer, even though the number of total collected oocytes (9.93 ± 9.1; 7.93 ± 5.38), MII oocytes (8.36 ± 7.64; 6.79 ± 4.63) and fertilized oocytes (5.25 ± 6.41; 3.91 ± 2.82), tended to be slightly superior in high vs moderate PA, there were no significant differences. In women with normal BMI, high PA was associated with a greater number of collected oocytes (p = 0.005), MII oocytes (p = 0.004) and fertilized oocytes (p = 0.007). Limitations, reasons for caution This study was performed in infertile women undergoing IVF/ICSI cycles. Hence, it is not possible to determine if PA has an impact on natural fertility. The self-administration of IPAQ has some disadvantages as subjectivity, memory bias or overestimation of PA. Subgroups’ sample size was small. Wider implications of the findings In general it does not seem that modifying short-term PA in women undergoing IVF could improve oocyte quality. However in some subgroups of patients (endometriosis, disovulation, normal-weight women), perhaps changes in PA could improve oocyte quality and ART success rates. More prospective clinical studies are needed. Trial registration number Not applicable

Gamma secretase inhibition: Effects on fertility and embryo-fetal development in rats

Avagacestat inhibits γ-secretase, a protease that cleaves the amyloid precursor protein (APP) to produce amyloid beta (Aβ). Aβ plaques, a predominant lesion in Alzheimer's patient's brain, is considered a mechanism driving neurodegeneration. As part of the nonclinical reproductive safety assessment, avagacestat effects on fertility and early embryonic development and embryo-fetal development were evaluated in rats. In the embryo-fetal development study, avagacestat was a selective developmental toxicant with dose-related increased fetal mortality, decreased fetal growth, and increased fetal malformations and variations (primarily affecting the axial and appendicular skeletal system) at ≥3 mg/kg/day. In the female fertility and early embryonic development study, avagacestat-related reductions in female fecundity at ≥5 mg/kg/day were attributed to impaired ovarian follicular development that was reflected in dose-dependent reductions in implantation sites, litter size, and gravid uterine weights. In the male fertility and early embryonic development study, avagacestat-related effects on reproduction could not be fully assessed because of low systemic exposures achieved due to extensive metabolism and clearance of the drug. The results obtained in these studies were consistent with pharmacologically mediated inhibition of γ-secretase and resulting inhibition of Notch processing and signaling that are key for embryonic development and ovary folliculogenesis. These findings are not considered a risk for late-onset AD where the patient population is ≥65 years old most with women who are post-menopausal. However, for treatment of early onset AD with a younger patient population, there are risks for reproductive or developmental toxicities with treatment with gamma secretase inhibitors like avagacestat.

Fertility and Developmental Toxicity Assessment in Rats and Rabbits with LY500307, a Selective Estrogen Receptor Beta (ERβ) Agonist

LY500307 is a selective estrogen receptor beta (ERβ) agonist that was developed for the treatment of benign prostatic hyperplasia. The in vitro functional selectivity of LY500307 for ERβ agonist activity is 32-fold above the activity at the alpha receptor (ERα). LY500307 was evaluated in a series of male (M) and female (F) rat fertility and rat and rabbit embryo-fetal development (EFD) studies, using 20 or 25 animals/group. LY500307 was administered daily by oral gavage starting 2 weeks (F) or 10 weeks (M) before mating, during cohabitation, until necropsy (M) or through gestation day (GD) 6 (F) in the fertility studies and from GD 6 to 17 (rats) or GD 7 to 19 (rabbits) in the EFD studies. Dosage levels of LY500307 ranged from 0.03 to 10 mg/kg/day for rats and from 1 to 25 mg/kg/day for rabbits. Fertility, estrous, maternal reproductive endpoints, conceptus viability, sperm parameters, organ weights, and histopathology were evaluated in the fertility studies. Maternal reproductive endpoints and fetal viability, weight, and morphology were evaluated in the EFD studies. Toxicokinetics were assessed in satellite animals. At 10 mg/kg/day in the male fertility study, findings included decreased body weight (BW); food consumption (FC); fertility, mating, and conception indices; sperm concentration; and reproductive tissue weight (associated with atrophic histologic changes). In the female fertility study, effects included decreased BW and FC at ≥0.3 mg/kg/day and persistent diestrus, delayed mating, and reduced fertility/conception indices at 3 mg/kg/day. In the rat EFD study, findings included decreased maternal BW and FC and increased incidences of adverse clinical signs, abortion, maternal mortality/moribundity, postimplantation loss, and fetal skeletal variations at 3 mg/kg/day. Effects in the rabbit EFD study were limited to decreases in maternal BW and FC at 25 mg/kg/day. In general, systemic maternal exposure increased proportionally with dosage in rats, but less than proportionally in rabbits. In conclusion, the no-observed adverse effect levels following LY500307 administration were 1 mg/kg/day for male rat fertility, 0.3 mg/kg/day for female rat fertility and EFD, and 25 mg/kg/day for rabbit EFD. Adverse reproductive and developmental effects only occurred at or above parentally toxic dosage levels and were considered predominantly due to off-target ERα effects.

Male and Female Fertility Assessment in the Cynomolgus Monkey Following Administration of ABT‐874, a Human Anti‐IL‐12/23p40 Monoclonal Antibody

ABT-874 is an anti-IL-12/23 monoclonal antibody that binds to the p40 subunit of human IL-12 and IL-23. As part of its preclinical safety assessment, studies were conducted to asses its potential effects on the reproductive system in male and female cynomolgus monkeys. Sexually mature male cynomolgus monkeys (n = 6/group) were administered once weekly subcutaneous doses of 0, 5, 25, or 100 mg/kg ABT-874 for 13 weeks. Four monkeys/group were necropsied at the end of the 13-week dosing period and two monkeys/group were necropsied following an 8-week recovery period. Endpoints assessed in these males included sperm parameters such as sperm count and morphology, male reproductive hormones, and testes histopathology. Sexually mature female cynomolgus monkeys (n = 6/group) were administered subcutaneous doses of 0, 5, 25, or 100 mg/kg/week ABT-874 for two menstrual cycles, and recovery was subsequently assessed in each of these animals over two menstrual cycles. Endpoints assessed in these females included menses and reproductive hormone levels. In both the male and female fertility studies, there were no unscheduled deaths and there was no evidence of toxicity. In male monkeys, there were no ABT-874-related effects on sperm count or motility, histopathology of the testes or effects on testosterone and inhibin B levels. In addition, menstrual cycle length, progesterone, 17ß-estradiol, and luteinizing hormone levels in female monkeys were comparable among control and ABT-874-treated groups. These results demonstrate that ABT-874 had no adverse effects on reproductive hormones or fertility parameters in male or female cynomolgus monkeys.

The Effects of Interleukin‐6 Signal Blockade on Fertility, Embryo‐fetal Development, and Immunization In vivo

Possible effects of interleukin-6 (IL-6) on reproductive performance, embryonal development, parturition, and postnatal development have been suggested based on protein/mRNA expression level of IL-6 in related organs, but less is known about functions of IL-6 signals in these areas. Following two different approaches have been employed to investigate the role of IL-6 signals in fertility and pre-/postnatal development: administration of a rat anti-mouse IL-6 receptor antibody, MR16-1, to mice as a neutralizing antibody system, and B6.129S2-Il6(tm1Kopf)/J (IL-6 knockout [KO]) mice as a KO system. By intravenously dosing 50 mg/kg of MR16-1 every 3 days, animals in male and female fertility studies and dams in a pre-/postnatal development study exhibited plasma MR16-1 concentrations much higher than the effective plasma concentration, indicating that MR16-1 exposure was sufficient to completely block IL-6 signals. The concentration of MR16-1 in the plasma of fetuses exceeded that in the plasma of pregnant animals, and MR16-1 concentration in milk was about one-fourth of that in plasma. Both the transient IL-6 signal blockade by MR16-1, and the constitutive IL-6 signal inhibition using IL-6 KO mice in a combined fertility and pre-/postnatal development study, revealed no biologically important effects on fertility, early embryonic development to implantation, or pre-/postnatal development, including IgG/IgM production by keyhole limpet hemocyanin sensitization. These results indicate that IL-6 signals have no unique, noncompensable roles in reproduction and development in the whole body system, although contributions of IL-6 in the signaling network appear to exist, as suggested by previously published investigations.

Collaborative work on evaluation of ovarian toxicity 1) Effects of 2- or 4-week repeated-dose administration and fertility studies with medroxyprogesterone acetate in female rats

As a part of the collaborative study to evaluate the relationship between histopathological changes of the ovary and functional changes in female fertility, 2- and 4-week repeated-dose toxicity studies and a female fertility study were conducted using female Crl:CD(SD) rats using a synthetic progestagen of medroxyprogesterone acetate (MPA) as a test compound. MPA was administered to female rats by gavage at 0, 0.4, 2.0 and 10 mg/kg/day for 2 and 4 weeks to assess the histopathological changes of ovaries and to pregnant rats at the same doses from 2 weeks prior to mating until day 7 of gestation to examine female fertility. The number of non-pregnant female rats with irregular estrous cycle increased in number and there was a decrease in weight of ovaries was observed at doses > or = 2.0 mg/kg in the 2- and 4-week-treatment groups. The histopathological examination revealed an increased number of large atretic follicles and decreases in currently formed corpora lutea and previously-formed large or small ones were observed at the same doses in the 2- and 4-week treatment groups. In female fertility study, the number of animals with an irregular estrous cycle and elongation of mean estrous cycle increased at 0.4 mg/kg, with no changes in fertility. A decreased number of copulating animals and a decreased gestation rate with low preimplantation loss were observed in the 2.0 mg/kg-treatment group and no copulation was observed in the group treated with 10 mg/kg. Based on these results, changes in fertility induced by MPA correlated well with histopathological changes of ovaries after 2 and 4 weeks of treatment, which suggests that a 2 weeks administration period is sufficient to detect ovarian toxicity of MPA with repeated dosing

Reproductive and Developmental Toxicity Evaluation of Desoris (LLL 3348) in Wistar Rats

Desoris (LLL 3348), a lyophilized aqueous extract prepared from the leaves of Argemone mexicana to treat chronic stable plaque-type psoriasis, was evaluated for reproductive (male and female fertility) and developmental toxicity in rats. Lrrp: Wistar rats were administered orally with LLL 3348 at dose levels of 0 (distilled water), 250, 500, and 1000 mg/kg b.wt, and the effects on reproductive parameters were assessed. Sperm parameters (motility, epididymal sperm concentration, testicular sperm head count, and sperm morphology), organ weight, and histology of the male reproductive system were evaluated in the male fertility study. Estrus cyclicity, corpora lutea, implantation sites, litter size at birth, fetal growth, development parameters up to weaning, and organ weight and histology of male and female reproductive systems were assessed in the female fertility study. There were no overt signs of toxicity noted in male and female reproduction parameters in rats up to 1000 mg/kg of LLL 3348 administration. There were no alterations in the male reproductive organ/system, sperm parameters, male and female fertility indices, embryonic development, and pre-wean developmental landmarks of pups. No gross and histological changes were observed in these studies. In a develop mental toxicity study, the test article was administered to pregnant females during gestation (5-19 days) and the fetuses were examined for external, visceral, and skeletal abnormalities. No toxic manifestation was revealed on caesarian section parameters, and no fetus anomalies/abnormalities were found. Therefore, it is concluded that LLL 3348 at the given dose did not produce any significant toxic effect in rats. The No Observed Adverse Effect Level (NOAEL) for male fertility, female fertility, and developmental toxicity studies was established as 1000 mg/kg in rats.

Collaborative work on evaluation of ovarian toxicity 2) Two- or four-week repeated dose studies and fertility study of mifepristone in female rats

In order to assess ovarian pathological changes and their relationship to changes in female fertility parameters, mifepristone, a progesterone receptor antagonist, was selected as the test article and was administered orally to female rats at dose levels of 0, 0.8, 4, 20 and 100 mg/kg for 2 or 4 weeks in repeated dose-toxicity studies and in a female fertility study at dose levels of 0, 0.8, 4 and 20 mg/kg from > 2 weeks before copulation to postcoital day 7. In the repeated dose toxicity studies, persistent estrus was seen in the vaginal smears, and multiple cysts in the ovaries at necropsy, increases in luteinized cysts and hypertrophy of previously formed corpora lutea were observed in the histopathological examination of ovaries in rats receiving 20 mg/kg or more for 2 or 4 weeks. In female fertility studies, persistent vaginal cornification was also observed at 20 mg/kg and the precoital interval was significantly shortened. All of the animals were completely infertile when dosed with 20 mg/kg during the post-coital period. An increase in pre-implantation losses was observed in the animals treated with 20 mg/kg during the pre-coital phase, while treatment with 4 mg/kg mifepristone during the post-coital phase induced an increase in post-implantation losses. These results suggested that a 2-week administration period would be sufficient to detect the ovarian toxicity of mifepristone in repeated dose toxicity study and the pathological findings in the ovaries would reflect the alterations in female reproductive endpoints in the female fertility study.

Collaborative work on evaluation of ovarian toxicity 12) Effects of 2- or 4-week repeated dose studies and fertility study of indomethacin in female rats

2-week and 4-week general toxicity studies of indomethacin, a nonselective inhibitor of cyclooxygenase 1 and 2, were performed using rats. A female fertility study was also conducted to compare the results to those of ovarian histopathological findings. The main purposes of the present studies are to assess whether a precise histopathological examination, taking the morphological changes the female reproductive organs undergo during each estrus phases into account, can evaluate toxicity to the ovaries, and to determine the optimal administration period for detecting ovarian toxicity. Indomethacin was administered on a daily basis to female Sprague-Dawley rats at doses of 0, 0.4, 1.3, or 4 mg/kg in the both the general toxicity studies and the female fertility study. In the general toxicity studies, unruptured follicles or luteinized cysts were observed histopathologically in the 4 mg/kg group in both the 2-week and 4-week studies. In addition, follicular cysts were found in the 4 mg/kg group in the 4-week study. Estrous cyclicity was not disturbed in both studies. There were no histopathological changes in the ovaries of the 1.3 mg/kg group in general toxicity studies. In the female fertility study, no toxic effects on female fertility parameters were detected in the 0.4 and 1.3 mg/kg group treated with indomethacin, but 8 of 10 rats in the 4 mg/kg group died or were sacrificed before completion of the dosing period. These results demonstrated that 2 weeks of indomethacin treatment is sufficient to detect unruptured follicles or luteinized cyst in the ovary. In addition, 4 weeks of dosing maybe required for induction of follicular cysts, although we could not clearly show that these histopathological changes would affect female fertility functions. These present studies suggest that a precise histopathological examination may be able to predict the effect of test articles on female reproductive functions.

Collaborative work on evaluation of ovarian toxicity 13) Two- or four-week repeated dose studies and fertility study of PPAR .ALPHA./.GAMMA. dual agonist in female rats

The main focus of this study was to determine the optimal dosing period in a repeated dose toxicity study based on toxic effects as assessed by ovarian morphological changes. To assess morphological and functional changes induced in the ovary by a peroxisome proliferator-activated receptor (PPAR) alpha/gamma dual agonist, the compound was administered to female rats at dose levels of 0, 4, 20, and 100 mg/kg/day in a repeated dose toxicity study for 2 or 4 weeks, and from 2 weeks prior to mating to Day 7 of pregnancy in a female fertility study. In the repeated dose toxicity study, an increase in atresia of large follicles, a decrease in corpora lutea, and an increase in stromal cells were observed in the treated groups. In addition, the granulosa cell exfoliations into antrum of large follicles and corpora lutea with retained oocyte are morphological characteristics induced by this compound, and they might be related with abnormal condition of ovulation. In the female fertility study, the pregnancy rate tended to decrease in the 100 mg/kg/day group. At necropsy, decreases in the number of corpora lutea, implantations and live embryos were noted in the 20 and 100 mg/kg/day group. No changes were observed in animals given 4 mg/kg/day. These findings indicated that histopathological changes in the ovary are important endpoints for evaluation of drugs inducing ovarian damage. In conclusion, a 2-week administration period is sufficient to detect ovarian toxicity of this test compound in the repeated dose toxicity study.

Collaborative work on evaluation of ovarian toxicity 5) Two- or four-week repeated-dose studies and fertility study of busulfan in female rats

Busulfan, an antineoplastic agent that targets small follicles (primordial and primary follicles), was given orally to female Sprague-Dawley rats (0, 0.1, 0.5, or 1.5 mg/kg/day; n = 10 in each group) for 2 or 4 weeks to assess the optimal administration period for detection of the toxic effects on ovarian morphology. Isolated ovaries were used for histopathological analysis and follicle counts. In addition, a female fertility study was conducted by giving the same dose levels of busulfan from 2 weeks before mating to day 7 of pregnancy to determine the non-observed-adverse-effect-level (NOAEL) for female reproduction. In the 2-week study, all rats treated with busulfan showed normal estrous cyclicity and no toxicological changes in weight or histopathology of the ovaries. In the 4-week study, a decrease in small follicles was found histopathologically in 1 rat, even at 0.5 mg/kg, and in 4 rats at 1.5 mg/kg. Proliferating cell nuclear antigen immunohistochemistry of the follicles confirmed the above decrease in number of small follicles at 1.5 mg/kg. In the female fertility study, increases in dead embryos and post-implantation loss were found in rats at 1.5 mg/kg. Taken together, the NOAELs were 1.5 mg/kg for reproductive performance and 0.5 mg/kg for early embryonic development. In conclusion, the present study indicates that a 4-week administration period and appropriate assessment, including careful histopathological analysis of stage-based follicles are needed to detect small follicle depletion in a general toxicity study used as a first-titer screen.

Collaborative work on evaluation of ovarian toxicity 6) Two- or four-week repeated-dose studies and fertility study of cisplatin in female rats

The main aim of the present study is to determine the optimal administration period of cisplatin with regards to its toxic effects on ovarian morphology in the repeated-dose toxicity study. Cisplatin was administered to female SD rats intraperitoneally once daily at dose levels of 0.25, 0.5, 1.0 and 2.0 mg/kg for 2 weeks, or at dose levels of 0.125, 0.25 and 0.5 mg/kg for 4 weeks in the repeated-dose toxicity study. In the female fertility study, 0.25, 0.5 and 1.0 mg/kg of cisplatin were administered in the same manner from 14 days prior to mating to Day 7 of gestation. In the repeated-dose toxicity study, a decrease in large follicle, an increase in atresia of medium and large follicles, and/or a decrease in currently formed corpus luteum were observed in animals receiving 1.0 and 2.0 mg/kg for 2 weeks, and decreases in small and/or large follicles and an increase in atresia of large follicle were observed in animals receiving 0.25 and 0.5 mg/kg for 4 weeks on the histopathological examination of the ovaries. In the female fertility study, the copulation and fertility indices in the animals receiving 1.0 mg/kg tended to be lower than those in the control animals. In conclusion, histopathological changes in the ovary that were attributable to cisplatin dosing were detected by detailed observation of the ovary in the 2-week study; and therefore, a 2-week administration period is sufficient to evaluate the ovarian toxicity of cisplatin.

Collaborative work on evaluation of ovarian toxicity 10) Two- or four-week repeated dose studies and fertility study of di-(2-ethylhexyl) phthalate (DEHP) in female rats

The main purpose of this collaborative work is to determine the optimal administration period required to detect toxic effects in evaluation of ovarian morphological changes in repeated-dose toxicity studies. To assess the morphological and functional changes induced in the ovaries by di-(2-ethylhexyl) phthalate (DEHP), two repeated-dose toxicity studies (repeated dose for 2 or 4 weeks) of DEHP administrated to female rats at dose levels of 0, 300, 1,000 and 3,000 mg/kg were conducted in collaboration with a female fertility study at the same dosages from 2 weeks prior to mating to Day 7 of pregnancy. Histopathology of the ovaries in both repeated-dose toxicity studies showed vacuolation of stromal cells in the groups receiving 300 mg/kg or more and an increase of large atretic follicles in groups at 1,000 mg/kg or more. In the 4-week study, a decrease in new corpora lutea was observed in the 3,000 mg/kg group. In the female fertility study, the 3,000 mg/kg group showed prolongation of the mean estrous cycle and irregular estrous cycles. Cesarean section revealed a decrease of pregnancy rate in the 3,000 mg/kg group. No effects on fertility or early embryonic development were found in groups at 1,000 mg/kg or less. These findings indicate that histopathological changes in the ovary are important endpoints for the evaluation of drugs which induce ovarian damage. In conclusion, for a repeated-dose toxicity study, a 2-week administration period is sufficient to detect ovarian toxicity caused by DEHP.

Collaborative work on evaluation of ovarian toxicity 9) Effects of 2- or 4-week repeated dose studies and fertility study of di(2-ethylhexyl)adipate (DEHA) in female rats

The present study was designed to confirm whether or not the ovarian toxicity of di(2-ethylhexyl)adipate (DEHA), which is known to have effects on female fertility, could be evaluated by the new method of histopathological examination of the ovaries in repeated dose toxicity. DEHA was orally administered to Crl:CD(SD) female rats at the doses of 0, 200, 1,000 and 2,000 mg/kg for 2 or 4 weeks in repeated dose toxicity study and for 2 weeks before mating, throughout mating and until Gestation Days 7 in female fertility. In the repeated dose toxicity studies, increase in atresia of large follicle, decrease in currently formed corpus luteum and follicular cyst were observed in the 1,000 mg/kg and above groups, suggesting that DEHA disturbed ovulation and large follicle growth. In the fertility study, a significant increase in mean estrus cycle length and post-implantation loss rate were observed in the 1,000 mg/kg and above groups, and a significant decrease in implantation rate and number of live embryos and a significant increase in pre-implantation loss rate were observed in the 2,000 mg/kg group. The histopathological changes of ovary observed in the repeated dose toxicity studies were correlated with the result that DEHA affected the estrus cycle in the female fertility study. In conclusion, a 2-week administration period is sufficient for detection of the ovarian toxicities following treatment with DEHA by new histopathological examination of the ovaries.

Collaborative work on evaluation of ovarian toxicity by repeated-dose and fertility studies in female rats

The National Institute of Health Sciences (NIHS) and 18 pharmaceutical companies of the Japan Pharmaceutical Manufacturers Association (JPMA) have conducted a validation study intended to evaluate whether a 2-week repeated general toxicity period with histopathological examination is sufficient to detect ovarian toxicity or not. The current repeated dose general toxicity study is considered to be insufficient in terms of evaluating female reproductive function due to a lack of evidence indicating that it is adequate. Evaluation of ovarian toxicity by comprehensive histopathological examination of the female reproductive organs based on the underlying morphology of a normal cycle of the reproductive tract including the ovary and additional immunohistochemical staining with proliferative cell nuclear antigen (PCNA) to identify small follicles may be a good tool to assess female reproductive function. In the collaborative study, 2- or 4-week repeated dose toxicity studies with ovarian histopathological examinations were conducted. A female fertility study was also conducted to compare the results with those of the ovarian histopathological findings. A total of 17 test substances were evaluated and categorized into hormone analogues, primordial follicle damaging agents, metabolite imbalance inducers, and endocrine imbalance inducers. Based on the results, ovarian toxicity could be detected by a careful histopatholgical examination. A 2-week dosing period may be sufficient for the evaluation of ovarian toxicity, except for cytotoxic compounds such as alkylating agents. The pathological findings of ovarian toxicity (decreases in follicles, increases in atretic follicles, increases in currently formed corpora lutea, etc) reflected the female fertility parameters (irregular estrous cycle, pre-implantation loss).

Collaborative work on evaluation of ovarian toxicity 8) Two- or four-week repeated-dose studies and fertility study of Anastrozole in female rats

The main focus of this study was to determine the optimal administration period in terms of toxic effects on ovarian morphological changes. To assess the morphological and functional changes induced by anastrozole in ovaries, the compound was administered to female rats at dose levels or 0, 0.01, 0.1 and 50 mg/kg for 2 or 4 weeks in the repeated dose toxicity study and at levels of 0, 0.01, 0.1 and 5 mg/kg from 2 weeks prior to mating to Day 7 or pregnancy in the female fertility study. In the repeated dose toxicity study, large abnormal atretic follicles, follicular cysts, a decrease in corpus luteum and depletion of developing corpus luteum were observed in the 1 and/or 50 mg/kg groups of both the 2-week and 4-week studies in a histopathological examination of the ovaries. In the female fertility study, the pregnancy rate was decreased in the 5 mg/kg group. Irregular estrous cycles, such as an extended cycle or no cycle, were observed in the 0.1 and 5 mg/kg groups. At necropsy, decreased numbers of implantations, corpora lutea and live fetuses were noted in the 1 and/or 5 mg/kg groups. Based on these findings, histopathological changes in the ovary are important endpoints for the evaluation of drugs inducing ovarian damage. We conclude that a 2-week administration period is sufficient to detect ovarian toxicity of anastrozole in a repeated dose toxicity study.

Collaborative work on evaluation of ovarian toxicity 3) Effects of 2- or 4- week repeated-dose toxicity and fertility studies with tamoxifen in female rats

To assess whether ovarian histopathological examination in repeated-dose rodent toxicity study could reliably anticipate toxic effects on female reproductive function and to assess whether ovarian change could be detected in a 2-week repeated-dose toxicity study, tamoxifen was administrated orally to female rats at 0.005, 0.03, or 0.2 mg/kg/day for 2 and 4 weeks in the repeated-dose toxicity studies, and for 2 weeks prior to cohabitation, during cohabitation, and through Gestation Day 7 in a female fertility study. The relationship between ovarian histopathological findings and fertility results was investigated. Findings at 0.03 and 0.2 mg/kg/day included decreases in body weight gains associated with decreases in food consumption, in 2- and 4-week repeated-dose toxicity studies and fertility study. The ovarian histopathological findings included increases in large atretic follicles, increases in interstitium cells and absence of newly-formed corpus lutea at 0.2 mg/kg/day in the 2-week study and at 0.03 and 0.2 mg/kg/day in the 4-week study. The treatment induced estrogenic and antiestrogenic reactions in the uterus, while mucinous degeneration was detected in the vagina. Effects on female fertility consisted primarily of disturbance of estrus cycle and decreases in numbers of pregnant rats which were considered to be related to ovarian histopathological changes. Based on these findings, ovarian histopathological evaluation in the repeated-dose toxicity study could anticipate the effects of tamoxifen on female fertility via ovarian dysfunction at slightly toxic doses, and 2-week treatment of tamoxifen at appropriate dose could be sufficient to detect ovarian toxicity by microscopic examination.

Collaborative work on evaluation of ovarian toxicity 15) Two- or four-week repeated-dose studies and fertility study of bromocriptine in female rats

The main focus of this study is to determine the optimal administration period concerning toxic effects on ovarian morphological changes in a repeated-dose toxicity study. To assess morphological and functional changes induced in the ovary by bromocriptine, the compound was administered to female rats at dose levels of 0, 0.08, 0.4 and 2 mg/kg for the 2- or 4-week repeated-dose toxicity study, and for the female fertility study from 2 weeks prior to mating to day 7 of gestation. In the 2-week repeated-dose toxicity study, increase of ovarian weights was observed at 2 mg/kg. In the 4-week repeated-dose toxicity study, ovarian weights were increased at 0.4 and 2 mg/kg. The number of corpora luteum was increased in the 0.4 and 2 mg/kg groups of the 2- and 4-week repeated-dose toxicity studies by histopathological examination of the ovaries. Bromocriptine did not affect estrous cyclicity in 2- and 4-week repeated dosing. In the female fertility study, although animals in any groups mated successfully, no females in 0.4 and 2 mg/kg groups were pregnant. There were no adverse effects on reproductive performance in the 0.08 mg/kg group. Based on these findings, the histopathological changes in the ovary are considered important parameters for evaluation of drugs including ovarian damage. We conclude that a 2-week administration period is sufficient to detect ovarian toxicity of bromocriptine in a repeated-dose toxicity study.

Collaborative work on evaluation of ovarian toxicity 7) Effects of 2- or 4- week repeated dose studies and fertility study of cyclophosphamide in female rats

The main focus of this study was to determine the optimal administration period concerning the toxic effects on ovarian morphological changes in the repeated dose toxicity study. In order to assess the morphological and functional changes induced in the ovary by cyclophosphamide (CP), the compound was administrated to female rats at dose levels of 0, 5, 10 and 20 mg/kg for the repeated dose toxicity study for 2 or 4 weeks, and at 0, 5, 10, and 20 mg/kg for the female fertility study from 2 weeks prior to mating to Day 7 of pregnancy. In the repeated dose toxicity study, increases in large sized atretic follicles, atrophy of corpora lutea were observed in the 20 mg/kg group in the 4-week study by the histopathological examination of the ovaries. There were no drug-related changes in the ovary in the 2-week study. In the female fertility study, the numbers of implantation were slightly decreased and the corpora lutea of pregnancy was not observed in the 20 mg/kg group. The dose-dependent increase in the incidence of post-implantation loss was observed, and no abnormalities were observed in the estrus cycle and mating in all treated groups. From these findings, the histopathological changes in the ovary are important endpoints for the evaluation of drug-induced ovarian damage as well as caesarean section. In conclusion, a 4-week administration period is sufficient to detect the ovarian toxicity of CP in the repeated dose toxicity study.