Persistent vaginal cornification

Biological state

The rodent cycle is subdivided in four subsequent phases, proestrous, estrous, metestrous (or diestrous 1) and diestrous (or dioestrous 2).  It is characterized by hormonal variation and consequent behavioural, morphological and physiological changes to the reproductive tract (ovary, uterus and vagina), describing these four phases.  Proestrous is the period during which pre-ovulatory development of the follicles takes place in the ovary. Estrous is the brief interval during which the female accepts the male and during which ovulation occurs. Next is metoestrous, the early luteal phase, followed by diestrous. The duration of the estrous cycle of rodent strains most commonly used in toxicology studies is typically 4 to 5 days (Goldman, 2007). For each phase of the cycle, the ovary, uterus and vagina have typical morphologic characteristics (Dixon, 2014).

Biological role in physiology

The keratinization of vaginal epithelial cells (vaginal cornification) that typically characterizes the day of estrous in the cycling rodents, is a consequence of the rising level of estradiol that peaks around midday on proestrous (Goldman, 2007).

Repeated failure of ovulation over successive days produces irregular cycles characterized by a persistent vaginal cornification (PVC). This state is called persistent estrous or constant estrous. Females in persistent estrous exhibit constant sexual receptivity (Westwood, 2008). The term persistent estrous (PE) in adult rats denotes failure of at least two consecutive estrous cycles as documented by cornified vaginal smears for 10 or more days (Singh, 2005).

Exposure to estrogenic compounds has commonly been reported to result in a persistent vaginal cornification (Goldman, 2007). As such, persistent vaginal cornification represents a good marker of prolonged E2 bioavailability in the uterus in regulatory studies.

It is noteworthy that persistent estrous is not equivalent to the true estrous phase. Indeed, at ovary level, estrous stage is characterized by the presence of new basophilic corpora lutea (CP) formed after the current ovulation and an absence of healthy (non-atretic) tertiary follicles. On the other end, a state of persistent estrous is characterized by ovaries with absence of basophilic corpora lutea, increased number of antral follicles and follicular cysts. Persistent estrous is comparable with the anovulatory cycles in women (Horvath 2004; Finch, 2014).

Regular cyclicity is one of the key parameters in assessment of female reproductive function in rodents. Parameters assessed for cyclicity:

• Number of cycling females
• Number of females with regular cycles
• Number of cycles
• Estrous cycle length
• Percentage of time spent in the various estrous cycle stages. Abnormal cycles were defined as one or more estrous cycles in the 21-day period with prolonged estrous (≥3 days) or prolonged diestrous (≥4 days) within a given cycle (Goldman, 2007).

Visual assessment

Visual observation of the vagina is the fastest method. It requires no special equipment and is best used when only proestrous or estrous stages need to be identified. It is non-invasive but the findings from this technique are observer-dependent. In the proestrous phase, the vaginal opening appears full, swollen and moist (Ajayi, 2020). The vaginal opening of mice in proestrous is characterized by swollen, moist, pink tissue. The opening is wide and there are often wrinkles or striations along the dorsal and ventral edges. As the mouse enters estrous, the vaginal opening becomes less pink, less moist, and less swollen (Byers, 2012).

Vaginal smear

Estrous cycle can be monitored in the rat and mouse by observing the changes in the vaginal smear cytology. It seems to be the most common technique used to determine the phases of the estrous cycle. It is non-invasive and relatively inexpensive (Goldman, 2007; Byer, 2012; Ajayi, 2020) and OECD guidelines (www.oecd.org).

The estrous phase shows abundant non-nucleate cornified epithelial cells. The cytoplasm is granular, and the cells are irregular in shape. Persistent estrous is characterized by cornified vaginal smears for 10 or more days.

Monitoring of estrous cyclicity is included in OECD test guidelines (TG 421: Reproduction/Developmental Toxicity Screening Test, 2016; TG 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 2016; TG 416: Two-Generation Reproduction Toxicity, 2001; TG 443: Extended One-Generation Reproductive Toxicity Study, 2018; and in USA EPA OCSPP 890.1450. Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal Female Rats Assay, 2011.

Histological examination of the reproductive organs

This technique is invasive and does not allow individual estrous cyclicity assessment. The minimal requirement for estrous staging evaluation by histology is the complete longitudinal sections of the vagina and cervix, transverse sections of the mid-portion of both uterine horns, and middle sections of both ovaries (Goldman 2007; Byers, 2012). Persistent estrous is characterized in ovary by the presence of numbers of ovarian follicular cysts (due to pre-ovulatory follicles losing the capacity to ovulate) absence of basophilic corpora lutea and decreased corpora lutea while uterine epithelium becomes tall columnar and vagina may show cornification (Westwood, 2008, Dixon, 2014, Shirai 2017, see also Fig. 16).

In OECD TG 443: For adult P and F1 females, a vaginal smear is examined on the day of necropsy to determine the stage of the estrous cycle and allow correlation with histopathology in reproductive organs.  Vaginal smear at necropsy is also required in OECD TG 408, OECD TG 421, OECD TG 422 while it is optional in OECD TG 407.

Other methods

Vaginal wall impedance and urine biochemistry are alternative methods. However, no standard values are available for commonly used laboratory animals (Ajayi, 2020).

The majority of the information comes from in vivo studies with rodents.

Regulatory Significance of the KE

While an evaluation of the estrous cycle in laboratory rodents can be a useful measure of the integrity of the hypothalamic-pituitary-ovarian reproductive axis, it can also serve as a way of ensuring that animals exhibiting abnormal cycling patterns are excluded from a study prior to exposure to a test compound. When incorporated as an adjunct to other endpoint measures, a determination of a female's cycling status can contribute important information about the nature of a toxicant insult to the reproductive system. In doing so, it can help to integrate the data into a more comprehensive mechanistic portrait of the effect, and in terms of risk assessment, may provide some indication of a toxicant's impact on human reproductive physiology. Significant evidence that the estrous cycle (or menstrual cycle in primates) has been disrupted should be considered an adverse effect (OECD, 2008). Included should be evidence of abnormal cycle length or pattern, ovulation failure, or abnormal menstruation (AOP 7).

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