Clinical Theriogenology 2022; 14: 70 Perinatal mortality in dogs and cats Christopher Premanandan,a Dalen Agnewb aCollege of Veterinary Medicine, The Ohio State University, Columbus, OH bCollege of Veterinary Medicine, Michigan State University, East Lansing, MI Abstract Canine and feline perinatal loss is a frequent and frustrating event for practitioners and breeders. Working on these cases can also be enigmatic and difficult for diagnostic pathologists to determine the cause of death. Typical conditions associated with canine perinatal loss include infectious disease, sepsis, pneumonia, congenital abnormalities, and trauma. Fading puppy syndrome is also a consideration. Currently, feline perinatal loss is not well characterized. Maternal health, a critical factor in perinatal losses, should also be assessed. Collecting an endometrial biopsy could constitute a critical part of this evaluation; however, more inves- tigation is needed to correlate endometrial lesions to fertility. Keywords: Abortion, fetal loss, dogs, cats, diagnostic pathology, perinatal mortality, necropsy Introduction Neonatal period in dogs is not well defined but is often re- ferred to as a period from birth to 30 days. Additionally, the early neonatal period is defined as the first 7 days of life and the late neonatal period is considered 1 - 4 weeks. Perinatal mortality can be defined as death that take place before first week of life in addition to stillbirth. Defined percentages of lit- ter loss in dogs are most frequently recorded in breeding col- onies. Of all mortalities 55.6% occurred before the first week of life (including stillbirth) with a perinatal death percentage of 11.9%.1 Perinatal loss in 10,810 litters in a Norwegian ken- nel club study was 8%.2 Only limited information is available regarding feline perinatal loss, and studies performed were less discriminatory and lacked clear distinction between early and late neonatal loss. However, the percent of stillbirth has been characterized. In 14 litters, 4.7% of the kittens were still- births.3 Analysis of a database comprised of French breeder litters described a rate of 8.5% over a 3-year period.4 Causes of perinatal death It is important to specify the types of infectious diseases that are related to stillbirth and the death of puppies and kittens within the first week of life. Since infectious diseases com- monly affect litters in the late neonatal period, confirmation of similar infectious agents as a cause of stillbirth or early neo- natal loss is more difficult.5 In addition, there is conflicting information regarding the incidence of perinatal loss due to infectious disease.2,6 Causes of perinatal loss in dogs Comments Fading puppy syndrome Pathogenesis unknown; refer to subheading below Bacterial pathogens Beta-hemolytic Streptococcus spp., Escherichia Coli, Brucella canis7,8 Viral pathogens Canine herpesvirus-1, canine adenovirus-1, canine parvovirus-2 Congenital defects Losses from 1.0 to 2.2%1,9 Parasitic pathogens Toxoplasma gondii10 Maternal infections Refer to bacterial and viral pathogens Maternal trauma N/A Dystocia N/A Many of the etiologies listed above are not limited to the peri- natal period, specifically, infectious agents that overlap with canine abortion and late neonatal loss. Causes of perinatal loss due to infectious disease are fairly well established despite the difficulty in detecting pathogens in many cases. However, many noninfectious causes are poor- ly characterized. Specifically, noninfectious placental disease is not well characterized in dogs and cats. Guidelines for the Clinical Theriogenology 2022; 14: 71 pathologic evaluation of human placenta are provided by the College of American Pathologists.11 These guidelines classify the causes into 3 major categories; maternal conditions, fetal conditions, and placental abnormalities. Maternal conditions include conditions such as placental abruption, systemic dis- orders at term, and severe oligoamnios. Stillbirth and perina- tal death are among the criteria of the fetal category. Placental conditions include thrombosis, umbilical cord torsions, and retroplacental hemorrhage. Placental abruption, hematoma and infarction are well-established causes of growth restriction and fetal death in humans.12 Placental abruption represents hemorrhage in the retroplacental region at the location of the basal plate. Abruption is also most commonly associated with this location; however, hemorrhage at the margin of the pla- cental disc is possible.13 Ischemic lesions can be categorized as fetal vascular malfor- mation or maternal vascular malformation. Fetal vascular malformation is typically associated with macroscopic ab- normalities such as umbilical cord torsion, true umbilical cord knots, or excessive umbilical cord lengths.14 These par- allels exist in horses and cattle; however, these are not well characterized or reported in dogs and cats. Maternal vascular malformation is related to the remodeling process that takes place in the maternal vasculature. Spiral arteries are a unique vascular arrangement in the primate placenta in which tro- phoblastic invasion results in modified blood flow to the fetal membrane. Ultimately, this results in less vascular resistance and increased blood flow to the placental disc. Abnormalities in this process can result in a myriad of lesions in the chorio- allantois including infarction, hypoplasia, and retroplacental hematoma formation.14 Infarction can result from both ma- ternal and fetal vascular malformation; however, maternal vascular malformation appears to be the most commonly as- sociated predisposing cause. Infarct affecting > 5% of the non- peripheral placental structure is worthy of consideration.14 Vascular abnormalities, particularly ischemia, have not been well characterized in canine and feline labyrinths. Authors have commonly observed coagulation necrosis adjacent to the marginal hematomas in normal neonates. However, ar- eas of coagulation necrosis and hemorrhage have also been observed in the middle region of the labyrinth in cases where the cause of fetal loss could not be determined from the anal- ysis of fetal tissue. The potential for acute or chronic placental disease induced by vascular events should be considered and investigated in dogs and cats. Fading puppy syndrome Respiratory disease is a common feature of what many breed- ers call ‘fading puppy syndrome,’ a frequent cause of death in neonates. Recent and ongoing research at the Michigan State University suggests that developmental lung disease is likely a major cause of these cases, similar to a condition in human pediatric medicine.15 Histologically, pulmonary dysplasia in- cludes underdeveloped alveoli, pulmonary artery medial hy- pertrophy, increased numbers of thin-walled venous profiles, and increased capillary profiles. The 3-D reconstructions of the affected lungs have vascular abnormalities suggestive of arteriovenous shunts. Continued research, including genetic analyses, is required to better understand this condition in dogs and humans and will require rigorous examination of cases of respiratory disease in puppies. Canine endometrial disease Similar to cattle and horses, a means to assess the health and quality of the endometrium while maintaining the animal’s fertility, is important. Endometrial biopsies have been the mainstay of endometrial diagnostics in horses for some time, and to a lesser extent in ruminants. However, this technique has been difficult to apply to the bitch for several reasons: 1. Zonary placentation and the regional nature of the placental sites make it difficult to extrapolate the find- ings from 1 endometrial sample to the entire surface of the uterus. 2. The size of the patient requires small instrumenta- tion, blind sampling, and advanced operator techni- cal skill. 3. The sensitivity of the endometrium to trauma during diestrus (sampling during this period may induce cys- tic endometrial hyperplasia). However, given these limitations, there is still value to iden- tify endometrial lesions in real-time. General procedure is as follows: sampling should ideally occur at the end of diestrus (progesterone is < 1 ng/ml), but it may be possible during anestrus or proestrus. Dogs are restrained manually (and se- dated if necessary). An endoscope with a 5-French rigid instru- ment port is inserted transvaginally and upon visualization of the cervix, a 5-French biopsy instrument is passed transcervi- cally into the uterus. Collection of at least of 3 separate sam- ples is suggested. Sanitizing the endoscope is not required be- tween samples. Samples can be fixed in 10% neutral buffered formalin, 4% paraformaldehyde, or Bouin’s solution, accord- ing to pathologist’s preference. Careful handling of very small and delicate samples is critical as they can easily be crushed or lost. A 26-gauge needle may help in removing tissue from the biopsy instrument. Some caution is necessary during anestrus, as the endometrium is so thin-walled that perforation of the uterus is very easy and the sample may be only adipose tissue. However, no negative sequela was noted from these events. Full-thickness endometrial samples can also be taken during a laparotomy. It is tempting to take these samples during a Caesarian section; however, these samples can be difficult to interpret as inflammation, necrosis, and cystic change are a normal part of mid to late involution, placental delivery, and involution and other changes are likely masked by these fea- tures. Lesions described as most prevalent in cases of canine subfertility (lack of pregnancy or fetal resorption) include chronic endometritis, cystic endometrial hyperplasia, and endometrial fibrosis.16-18 Endometritis and fibrosis were not described during the immediate postpartum period in a study of 98 bitches at various stages of involution.19 The relative val- ue of biopsies taken during diestrus versus other stages of the estrous cycle has not been determined. However, since canine endometrium morphology undergoes a variety of histological changes during the estrous cycle, evaluation by a pathologist familiar with canine endometrium is advisable. Clinical Theriogenology 2022; 14: 72 Pyometra was not detected in transcervical endometrial bi- opsies; however, fibrosis, cystic endometrial hyperplasia, and inflammation (equal to the full thickness sections) were ob- served in endometrial samples obtained after ovariohysterec- tomy.20 All samples with pyometra were from diestrous ani- mals. Therefore, it is more likely that the sampling procedure itself induced pyometra. However, authors have identified multiple cases of pyometra and endometritis using transcer- vical endometrial biopsies. Whereas additional studies are needed, transcervical endometrial biopsies offer additional diagnostic information for bitch infertility, including repeated abortions. Feline endometrial disease Although there are many similarities between canine and feline placentation grossly, substantial variations can be ob- served microscopically, primarily in the chorionic labyrinth. Feline placental site involution studies are very limited and consequently it is difficult to elucidate endometrial associa- tion in subfertility. Although it is tempting to apply the knowl- edge that we have gleaned from bitch endometrium to queen, the endometrial lesions associated with feline infertility has to be explored. A systematic study of the lesions that affect feline endometrium and subsequent correlation of these lesions to subfertile queens would be an ideal starting point. Conclusion Investigation of canine and feline perinatal loss requires col- laboration and cooperation among the owner, clinician, and pathologist as the determining specific causes can be difficult. Perhaps managing expectations is a critical aspect of the pro- cess. Majority of investigations often result in negative histo- logical and ancillary diagnostic tests. However, the value of excluding infectious, particularly transmissible, causes of the perinatal death is often overlooked in this process. Diagnos- tic testing for infectious agents is well established and readily available in most commercial and state diagnostic laborato- ries. Critical investigations remain for noninfectious causes of death that may involve placental insufficiency and endome- trial disease. Additionally, further investigation of potential causes of ‘fading puppy syndrome’ is necessary as the patho- genesis of this condition is poorly understood. Conflict of interest Authors have no conflicts of interest. No funding was received for this publication. References 1. Potkay S, Bacher JD: Morbidity and mortality in a closed foxhound breeding colony. Lab Anim Sci 1977;27:78-84. 2. Tønnessen R, Borge KS, Nødtvedt A, et al: Canine perinatal mortality: a cohort study of 22 breeds. Theriogenology 2012;77:1788- 1801. 3. Root MV, Johnston SD, Olson PN: Estrous length, pregnancy rate, gestation and parturition lengths, litter size, and juvenile mortality in the domestic cat. J Am Anim Hosp Assoc 1995;31:429-433. 4. Fournier A, Masson M, Corbière F, et al: Epidemiological analysis of reproductive performances and kitten mortality rates in 5,303 purebred queens of 45 different breeds and 28,065 kittens in France. Reprod Domest Anim 2017;52(Suppl 2):153-157. 5. Sturgess K: Infectious diseases of young puppies and kittens. In: Simpson GM, England GCW, Harvey M: editors. Manual of Small Animal Reproduction and Neonatology. British Small Animal Veterinary Association: 1998. p. 159-166. 6. Lawler DF: Care and diseases of neonatal puppies and kittens. In: Kirk’s Current Veterinary Therapy X, Philadelphia; WB Saunders:1989. p.1325-1333. 7. Jones RL: Special considerations for appropriate antimicrobial therapy in neonates. Vet Clin North Am Small Anim Pract 1987:17:577-620. 8. Vela AI, Falsen E, Simarro I, et al: Neonatal mortality in puppies due to bacteremia by Streptococcus dysgalactiae subsp. dysgalactiae. J Clin Microbiol 2006;44:666-668. 9. Andersen AC Puppy production to the weaning age. J Am Vet Med Assoc 1957;130:151-158. 10. Bresciani KD, Costa AJ, Toniollo GH, et al: Transplacental transmission of Toxoplasma gondii in reinfected pregnant female canines. Parasitol Res 2009;104:1213-1217. 11. Langston C, Kaplan C, Macpherson T, et al: Practice guideline for examination of the placenta: developed by the Placental Pathology Practice Guideline Development Task Force of the College of American Pathologists. Arch Pathol Lab Med 1997;121:449-476. 12. Aurioles-Garibay A, Hernandez-Andrade E, Romero R, et al: Prenatal diagnosis of a placental infarction hematoma associated with fetal growth restriction, preeclampsia and fetal death: clinicopathological correlation. Fetal Diagn Ther 2014;36:154-161. 13. Mittal N, Byard RW, Dahlstrom JE: A practical guide to placental examination for forensic pathologists [published correction appears in Forensic Sci Med Pathol 2020 Jan 8]. Forensic Sci Med Pathol 2020;16:295-312. 14. Heider A: Fetal vascular malperfusion. Arch Pathol Lab Med 2017;14:1484-1489. 15. Tsoi, M, Frantz, B, Galambos, C, et al: The use of three dimensional images reconstruction to characterize lung vascular abnormalities in canine developmental lung disease. American College of Veterinary Pathology Annual Conference. 2019. 16. Fontaine E, Levy X, Grellet A, et al: Diagnosis of endometritis in the bitch: a new approach. Reprod Domest Anim 2009;44(Suppl 2):196-199. 17. Mir F, Fontaine E, Albaric O, et al: Findings in uterine biopsies obtained by laparotomy from bitches with unexplained infertility or pregnancy loss: an observational study. Theriogenology 2013;79:312- 322. 18. Gifford AT, Scarlett JM, Schlafer DH: Histopathologic findings in uterine biopsy sample from subfertile bitches: 399 cases (1990-2005). J Am Vet Med Assoc 2014;244:180-186. 19. Al-Bassam MA, Thomson RG, O’Donnell L: Normal postpartum involution of the uterus in the dog. Can J Comp Med 1981;45:217- 232. 20. Christensen BW, Schlafer DH, Agnew DW, et al: Diagnostic value of transcervical endometrial biopsies in domestic dogs compared with full-thickness uterine sections. Reprod Domest Anim 2012;47(Suppl 6):342-346.