Vaginal Septa Caused Atresia with Hydrometrocolpos Syndrome in BALB/c Mice

Vaginal septum and atresia are rare reproductive system defects of female mammals (Chang et al., 2013; Kim et al., 2017; Destro et al., 2018; Altinçekiçet_al2019). Published reports considered that the two defects were different genetic diseases, while underlying correlation between these two diseases remain unclear (Wu et al., 2010; Lezmi et al., 2011; Murata et al., 2015; Zhao et al., 2016; Xie et al., 2017). HMCPS was found in human congenital atresia, McCusco-Coffman syndrome, characterized by hydrometra, mucometra and hematocolpos with tortuous uterus and distended cervix (Lenartowicz et al., 2010; Lueth and Wood, 2014; Sharma et al., 2015; Destro et al., 2018). PCOS is a complex endocrine and metabolic disorder, which is characterized by chronic anovulation (dysfunction or loss of ovulation) and hyperandrogenism, usually mentioned in gynecological cases (Lenartowicz et al., 2010; Nakane and Biesecker, 2010; Vilanova-Sanchez et al., 2019).
       
Here, we report that vaginal septa induced spontaneous atresia as the mice matured and then progressively developed into HMCPS in our inbred female BALB/c mice, which were confirmed with anatomical, hematological, pathological, endocrinological and imaging investigations. Surgical intervention adopted on vaginal atresia mice successfully prevented further deterioration.

Laboratory animals and care
 
Our inbred colony of BALB/c mice was introduced in 2012 from National Rodent Experimental Animal Seed Center (Shanghai, China) and then breeding in the SPF (Specific Pathogen Free) barrier system of Gannan Medical University. The temperature (23±2°C), humidity (50%±10%) and light-dark cycle (10: 14h) in the SPF environment were kept constant. Mice in the barrier were fed with the standard sterile mouse food (Keli Xieao, China) and purified water filtered by American Millipore Water Purifier. Their bedding and cage were sterilized via high-pressure boiler (Xin-hua, China).
 
Animal ethics
 
All the experiments conducted on the mice were complied with the Chinese Guidelines for the examination of evidence of Laboratory Animal Welfare GB/T 35892-2018 and approved by the Ethics Committee of Gannan University.
 
Grouping
 
According to the abnormality of mice’s vagina, animals were divided in three groups as follows:
 
Imperforate vagina with septum (IVS) group
 
In this group, all mice showed vaginal septa before the closing of vagina and they were all sacrificed at 26-30 weeks old.
 
Opening vagina with septum (OVS) group
 
This group included the mice that observed with vaginal septa while keeping opening orifices on the septa. They were all sacrificed at 8-12 weeks old after their opening vagina period (onset of mice’s puberty).
 
No-septum in vagina (NSV) group
 
This was a control group. Animals were sacrificed at 26-30 weeks and 8-12 weeks old keep pace with the IVS or OVS case group.
 
Case history, surgical operation and findings
 
The first 2 IVS mice captured our attention with inflated abdomen and persistent infertility at their 28-week in the 11^th inbred generation. There was lots of light-yellow translucent liquid blocked in vagina and uteri. Unfortunately, these 2 mice remained infertile after postoperative anti-inflammatory and estrogen-mediated intervention. In order to exclude other unfavorable factors that may cause infertility, more stringent examinations on copulatory plugs were conducted. The other 24 mice were initially diagnosed with vaginal septa after weaning and 8 of the 24 mice’s vagina turned into imperforated vagina after their puberty. Herein, the 8 mice with atresia were allotted into IVS group and the other 16 mice with opening vagina were classified into OVS group. The 8 IVS mice were equally divided into two intra-group: 4 IVS mice were kept until they were 26-30 weeks old and the vaginal septa of the other 4 mice were removed with reported surgical intervention when they were 8-12 weeks old (Altinçekiçet_al2019; Chang et al., 2013; Vilanova-Sanchez et al., 2019; Wang et al., 2019). At the end, we found these 4 IVS mice in intervention group all restored their fertility through operation.
 
Image examination
 
B-type ultrasonic imaging was performed by DP-50VET (Mindray, China) and Direct digital (DR) X-ray photo was taken on the X-ray machine (Yuanzhi, China), The examination of vaginal septa was initially screened according to the reported method (Chang et al., 2013) and confirmed with anatomical microscope (Olympus, Japan). H.E. stained sections were photographed under inverted biological microscope (Leica, Germany).
 
Blood routine analysis, Serum biochemical and ELISA Tests
 
Blood was collected from eyes of mice. And euthanasia was completed by cervical dislocation after isoflurane (YiPin, China) anesthesia. Blood routine analysis was performed on SYSMEX-XN2000 blood analyzer (sysmex Kobe, Japan) at the first affiliated hospital of Gannan Medical University. And serum biochemical analysis was conducted on Roche cobas 8000 automatic biochemical analyzer in the hospital also. ELISA Tests were finished on enzyme marker (Epoch-Biotek, USA) with commercial kits (ShuangYing, China).
 
Sample collection and pathological examination
 
Necropsy was performed on each mouse and internal organs were weighed by scale and inspected by licensed veterinarian. Organs from FRS such as cervix, vagina, uterus and ovaries were embedded into paraffin, then sectioned and stained with hematoxylin-eosin (HandE) as the methods of references (Sharma et al., 2015; Kim et al., 2017; Ahuja et al., 2018). Electronic scales with an accuracy of 0.01g was used for weighing and vernier calipers was used to measure lengths and diameters with an accuracy of 0.02mm.
 
Statistical analysis
 
One-way ANOVA was used to compare variances between groups. When the P value was greater than 0.05, pairwise comparison between groups were conducted with the least significance difference (LSD) method was used for, assuming variance were homogeneous.

Different kinds of dorsoventral septa were observed in vagina (Fig 1A-E), which were closely related to the amenorrhea and infertility (Gearhart et al., 2004). The fluid would drain out of the vaginal duct smoothly in the normal mice, even when the vulva is swelling around the canal (Fig 1F). The vaginal orifice with septum seems to be jammed with mucous debris (Fig 1C). Some yellow, oily liquid was taken out from the vagina of an IVS mouse during the surgical operation after she was anesthetized (Fig 1D). Once the vaginal orifice was blocked by the menstrual discharge with the septa, much yellow liquid was produced and accumulated in the cervix, then the secondary HMCPS occurred and the draining baffled. When the IVS mice were 26-30 weeks old, all of them demonstrated secondary HMCPS. The HMCPS is characterized by extremely distended uterus and cervix (Fig 2A-C). As shown in Fig 2A, large number of occupying sinuous cable-like shadows appeared in the abdomen. And according to B-ultrasonography test of the uterus, a large area of dark fluid was observed in the uterus (Fig 2C).
 

 

       
In routine blood examination, there is few significant difference among IVS, OVS and NSV group, except NEUT, MCH, MCHC and NRBC%. The number of white blood cells (WBC) showed no significant difference between groups and all floated within the normal range. However, the number of neutrophils (NEUT) exhibited significant difference (P<0.05) between OVS and NSV group and extreme significant difference (P<0.01) between IVS and NSV group (Table 1). This indicates that the vaginal septum plays a role as mildly inflammatory stimulus before the closing of vagina. Once the atresia and HMCPS occurs, numerous exfoliated endometrium and intrauterine effusion become inflammatory stimulus which prompt neutrophils and neutrophil ratio increased. The number of red blood cells (RBC) showed no significant difference and were in the normal range, which means the numerous exfoliated endometrium and intrauterine effusion would not result into the loss of red blood cells. The decrease (P<0.01 or P<0.05) of value MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration) and NRBC% (nucleated red blood cell) and the rise of RDWCV% (red blood cell volume distribution width coefficient of variation) means the septum might give rise to iron-deficiency anemia, especially when the atresia and HMCPS occurs.
 

       
In the serum examination (Table 2-3), when IVS was compared with NSV (26-30 weeks old) and while OVS is paralleled with NSV (8-12 weeks old), the triglycerides (TG) and LDL (low-density lipoprotein cholesterol) items reveal extremely significant (P<0.01) between IVS and NSV (26-30 weeks old). The value of TP (total protein), ALB (albumin), CHO (cholesterol), HDL (high-density lipoprotein cholesterol), LDL (low-density lipoprotein), VLDL (very low-density lipoprotein) were extreme significant (P<0.01) or significant (P<0.05) different when OVS is paralleled with NSV (8-12 weeks old) and the TG of OVS was higher than NSV (8-12 weeks old). Therefore, it can be concluded that the vaginal septum promotes the chance of occurrence of atresia and HMCPS and PCOS could increase the metabolism of protein and fat before the closing of vagina. In the endocrinological analysis, there were 9 items including FSH, LH, PRL, E2, P, T, AZP Ab (Anti-zona pellucida antibody) and AOA (anti-ovarian antibody) tested by ELISA method (Table 3). The result of AZP Ab and AOA exclude the possibility of immune infertility in vaginal septum and atresia in mice. The indices of PRL (Prolactin) testify that there is no evidence that the hyperprolactinemia have relationship with the amenorrhea due to vaginal septum and atresia in mice. The data of FSH, LH (luteinizing hormone), P (Progesterone), T and PRL in IVS all show a downward tendency compared with NSV (26-30 weeks old), especially IVS mice’s FSH (Follicle Stimulating Hormone) which decreased nearly a half of NSV. Rather, the E2 (Estrogen) shows slight rise and the Aromatase, which is used to increase estrogen through aromatization from T (Testosterone), demonstrated a double increase in two-comparison between IVS and NSV (26-30 weeks old).
 

 

       
In the comparison of organ indexes between different groups based on weight or length, we found that the FRS of IVS (26-30 weeks old) significantly swelled (Table 4). All the FRS organ indexes, including length of uterus, diameter of uterus, weight of FRS, Ratio of (FRS (g) /Body (g)) of IVS (26-30 weeks old) were significantly increased (P=0.00: P<0.01) compared with NSV (26-30 weeks old). The Ratio of (FRS (g) /Body (g)) of IVS mice was much higher (about 7 times) than the NSV mice (Table 4). And we observed that the weight and sizes of FRS organs of OVS mice with varying degrees swelled (Table 4). These data suggested that the HMCPS plagued the IVS mice and even the OVS mice (Fig 2). The individual number led to the mild non-significant difference in FRS organ indexes between OVS and NSV group (8-12 weeks old).
 
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Finally, as is shown in Fig 3A, ovarian lesions are more serious in IVS mice in addition to uterine hydrops. The pathological section of ovaries demonstrated obviously excessive vacuoles degeneration of the corpus luteum. Mature follicles capable of fertilization is scarce. The cortical and medullary structures are indistinct in Fig 3A. It suggested that the primordial follicle differentiation in IVS was partially inhibited. The primordial follicles (type I follicles) proliferated and large amount of the secondary follicles and luminal follicles reduced in IVS’ ovaries (Lenartowicz et al., 2010; Sreejalekshmi et al., 2016).
 

       
In addition, as is shown in Fig 3B, the HE staining of the fused tip’s transection in urogenital sinus with the end of vagina verified that these atresia was derived from incompletely closed vagina with septa (OVS).

In this paper, we observed 26 BALB/c mice with vaginal septum and kinds of examinations were conducted. Investigations indicated that persistent effusion in uteri and long-term accumulation of toxins in IVS mice with HMCPS promoted the earlier apoptosis of secondary oocyte and stimulated numerous corpus luteum and albicans, led to numerous long-term residues vacuoles enclosed by epithelial fibers and eventually developed into PCOS.

This work was financed by Innovation Special Fund Project for Graduate Student in Jiang Xi Province (YC2019-B058). This work was supported by Foundation of Gannan Medical University (YB201822 and ZD201822), Doctoral Startup Foundation of Gannan Medical University (QD201802) and Instructional Technology Program of Ganzhou (GZ2018ZSF003).