Chlamydia trachomatis infection

Chlamydia trachomatis is an intracellular bacterial pathogen and a major cause of genital and eye disease. It comprises three human biovars. One causes trachoma, the most common form of blindness worldwide, and particularly prevalent in Africa. A second is the cause of lymphogranuloma venereum, a sexually transmitted infection (STI), mainly of gay men, in the developed world. The third biovar, consisting of serovars D to K, is the topic of this monograph. It is a major cause of PID, EP and TFI in women, epididymitis in men, and neonatal conjunctivitis and neonatal pneumonia in newborns.^3,28,63,64

Chlamydia trachomatis is transmitted by oral, vaginal or anal sex, and can also be transmitted from mother to newborn during a vaginal delivery.^3,63,64 Diagnosis is generally based on nucleic acid amplification tests (NAATs) carried out on cervical or vaginal swabs in women, urethral swabs in men or on urine samples.^3,63,64 NAAT has largely replaced culture or enzyme immunoassays for diagnostic testing.^3,63,64 However, culture played a key role in earlier epidemiological studies, and the lower sensitivity of culture, 60–80%,^64,65 needs to be taken into account when interpreting the earlier research results.

The infection can be treated effectively by antibiotics, such as azithromycin or doxycycline, but it is often asymptomatic, especially in women.^3,63,64 Re-infection by the same partner is an important issue in prevention: partner notification rates can be improved and risks of re-infection reduced by providing index cases with prescriptions intended for their partners, or with the medication itself.^66–71

Chlamydia trachomatis infection

Approximately 75% of incident infections in women are asymptomatic.⁷³ Although studies of the duration of asymptomatic infection have produced a wide range of estimates – between 1 and 18 months (see Chapter 4) – it is evident that the infection can clear spontaneously in the absence of treatment as a consequence of the adaptive and innate immune responses.^38,74

The risk of transmission of infection from one episode of sexual intercourse is estimated to be between 10% and 20%.^75,76 Given the high sensitivity of current NAATs for detecting CT,^8–10 and the fact that non-host DNA and sperm can be recovered from the female genital tract up to 7 days following sexual intercourse, this strongly implies that at least some women who test CT positive (CT+) are probably only passively infected.^77,78 This is consistent with the observations of Joyner et al.⁷⁹ and Geisler et al.⁸⁰ that a proportion of women testing CT+ without treatment become CT NAAT-negative within 2 weeks.

Prevalence of CT can be studied by population surveys, using NAATs on genitourinary specimens. As with most STIs, incidence and prevalence are highest among young people, and decline steeply with age.⁸¹ Infection is more common among the more sexually active individuals, with the greatest number of sexual partners.^8–10,81

Definition of pelvic inflammatory disease

Pelvic inflammatory disease comprises a spectrum of upper genital tract inflammatory disorders among women, which includes any combination of endometritis, salpingitis, tubo-ovarian abscess and pelvic peritonitis.⁸² The principal clinical and economic significance of CT lies in the fact that it is a causal agent of PID and the further damage to reproductive health that follows from salpingitis.^33,83,84 Confusion can arise with the term PID. Historically, it was often used interchangeably with the term ‘acute salpingitis’ as detected at laparoscopy, which, historically, was the gold standard for diagnosing PID.^85,86 More recently endometritis, which is associated with salpingitis but can be detected by endometrial sampling, has been equated with PID because of the unacceptability of performing laparoscopy on large numbers of women with minimal symptoms.^85–87

Pelvic inflammatory disease is not easy to diagnose and the criteria for a clinical diagnosis of PID have changed over time.³³ In the original Lund studies³³ the minimum criteria for diagnosis were: (1) lower abdominal or pelvic pain of < 3 weeks; (2) a purulent vaginal discharge diagnosed on microscopy or painful intermenstrual bleeding; and (3) increased motion tenderness of the uterus and adnexa on bimanual examination. In the UK national PID guideline 2011,⁸³ recent onset of lower abdominal pain in association with local tenderness on bimanual examination is now considered sufficient to establish a diagnosis and initiate treatment. This is because it is now recognised that many women with salpingitis have subtle or mild symptoms.^63,83 Even when present, the clinical symptoms and signs of PID lack sensitivity and specificity for detecting women with salpingitis.⁸³ Because of the difficulty of diagnosis and the potential for damage to the reproductive health of women (even by apparently mild PID) and the benefit of early antimicrobial therapy, health-care providers are now advised to maintain a low threshold for the diagnosis of PID.⁸³

Laparoscopic diagnosis of PID, which identifies women with salpingitis, is no longer undertaken in women with a clinical diagnosis of PID. However, clinical information can be used to classify PID as ‘possible’, ‘probable’ and ‘definite’ PID, based on Hager’s criteria.^88,89 Table 1 details the clinical definitions adopted by Taylor-Robinson et al.,⁸⁸ based on a modified version of the Hager criteria, and which were used to estimate the proportion of women with salpingitis following a clinical diagnosis of PID. This classification is often used in clinical trials such as POPI, and in studies of patient data such as the GPRD database, although in recent work using GPRD ‘probable’ PID includes chronic disease as well.^19,88,90 As the probability that patients diagnosed with PID based on clinical presentation have salpingitis decreases, the likelihood of other diagnoses, such as endometriosis, irritable bowel syndrome and functional pain, increases.

TABLE 1

Definition of clinical diagnosis of PID

For the purposes of this report we define clinically diagnosed PID as women with ‘probable’ and ‘definite’ diagnostic criteria. Women with ‘possible’ diagnostic criteria may be treated for PID depending on to where they present, which is probably less likely in primary care compared with departments of genitourinary medicine (GUM) as there is greater expertise in managing PID in the latter setting. Diagnostic thresholds have changed over time as the role of PID in reproductive damage has been recognised. Accordingly, the proportion of women with PID regarded as having ‘probable/definite’ or ‘possible’ PID diagnostic criteria is likely to have increased over time, with fewer women remaining symptomatic and undiagnosed.^19,90 This probably began in the mid to late 1990s in the UK.

Infections associated with pelvic inflammatory disease

Pelvic inflammatory disease is not exclusively caused by CT, with about 30–40% of PID cases being CT+^37,82 (see Chapter 7). A range of STIs have been implicated, particularly gonorrhoea and, more recently, Mycoplasma genitalium.^63,83,91 It is also likely that PID can be caused by micro-organisms associated with bacterial vaginosis (BV), which is commonly present in women with PID.^63,83,92 These micro-organisms include Gardnerella vaginalis and anaerobes (including Prevotella, Atopobium and Leptotrichia),^83,93 which are probably sexually transmissible but would not be classed as STIs.^92,93 Anaerobes are isolated more often from women with severe PID. As a consequence, broad-spectrum antibiotic therapy is recommended for PID to cover Neisseria gonorrhoeae, CT, and a variety of aerobic and anaerobic bacteria commonly isolated from the upper genital tract in women with PID.⁸³

Bacterial vaginosis is a common condition affecting women of reproductive age.^94,95 It is a condition characterised by vaginal flora imbalance, in which the normal plentiful lactobacillus is scarce and other anaerobic bacteria abundant.^94–96 It is present in between 12% and 25% of women.^94,95 Women are usually asymptomatic and if symptoms are present these consist of vaginal discharge with or without an odour.⁶³ Only some BV-associated bacteria have been associated with PID, and it remains to be established whether or not these bacteria are indeed causally implicated in reproductive damage.⁶³

Undiagnosed pelvic inflammatory disease and asymptomatic pelvic inflammatory disease

Over 50% of PID episodes in the UK are treated in primary care, but it is also treated in STI clinics and in hospital. Data from the GPRD, Hospital Episode Statistics (HES), and KC-60 returns from STI clinics for 2002 are shown in Table 2, which is based on the ‘definite/probable PID’ definition.⁹⁰ We have assumed that women admitted for inpatient treatment of PID in the HES data, and women diagnosed at departments of GUM by clinicians trained in diagnosing PID, are ‘probable’ or ‘definite’ cases. An unknown proportion of diagnosed PID episodes meeting these definitions will be recorded under more than one of these locations, although the study by Nicholson et al.⁹⁷ using the GPRD database suggests that approximately one-third of cases of probable or definite PID diagnosed in primary care have been treated elsewhere.

TABLE 2

Number of incident cases of PID in England, 2002

These figures on diagnosed PID incidence certainly underestimate the true incidence of PID episodes. It is widely accepted that a relatively high proportion of PID is undiagnosed. This is inferred from historical studies of TFI. PID is considered to be a necessary precursor of TFI,⁹⁸ and yet a high proportion of women with TFI have no history of clinical PID.^99–102 Wolner-Hanssen¹⁰³ found that only 34% of TFI cases reported a previous diagnosis of PID. However, only 11% reported never having had clinical symptoms.¹⁰³ The implication is that a large proportion of the PID that causes TFI is undiagnosed, but it is relatively unusual for PID which causes reproductive damage to be completely asymptomatic. We therefore distinguish diagnosed PID, by which we mean PID meeting the ‘probable/definite’ criterion, undiagnosed symptomatic PID and asymptomatic or silent PID (see Chapter 7).

Therefore, even if we maintain a single diagnostic criteria, such as ‘probable/definite’, across all studies, and these definitions were consistently applied – which we know is not the case – it would still be the case that the proportions of PID that would be ascertained would depend on the study design. For example, the routine PID statistics in Table 2 depend on patient self-referral, whereas in a prospective study of women attending a STI clinic, or in the context of a screening trial, a higher proportion of PID meeting the same definition may come to light (see Chapter 7).

Current guidance on pelvic inflammatory disease management

Recognising the difficulty of achieving a definitive diagnosis, the British Association for Sexual Health and HIV (BASHH) guidelines⁸³ emphasise a low threshold for empiric treatment, to lower the risk of subsequent EP and infertility. The same advice is seen in primary care guidelines and the National Institute for Health and Care Excellence (NICE) Clinical Knowledge Summaries.^104,105 However, the latter cites multiple partners, a recent new partner, young age and previous history of PID or a STI as risk factors for PID. The NICE Clinical Knowledge Summary¹⁰⁴ explicitly states that ‘PID is almost always due to a sexually transmitted disease’. This contrasts somewhat with BASHH guidelines,⁸³ which acknowledge that only 25% of cases of can be accounted for by CT or N. gonorrhoeae. US guidelines highlight young age as a risk factor but avoid citing any behavioural risk factors.⁶³ All guidance advocates early use of broad-spectrum antibiotics.

Age profiles of Chlamydia trachomatis and pelvic inflammatory disease

In this section we briefly compare the age profiles of CT and PID, to draw attention to an important, but generally overlooked, issue in the natural history of CT. Estimates of CT prevalence are approximately fourfold higher in women aged 16–24 years than women aged 25–44 years.¹⁰⁶ The age profile of incident PID is very different. HES show an increasing trend in PID incidence rates with age (see Table 2). Evidence from the GPRD shows incidence rates that are higher in the age group 20–24 years, but the difference in PID incidence rates across ages is far less than the difference in chlamydia prevalence. The age profile in PID presenting in STI clinics is more similar to the age profile of incident CT, in that there is a relatively high proportion of young women. However, of the three sources, these make up the smallest proportion of diagnosed cases.

There are three plausible contributory reasons for these discrepancies in age profile. First, the PID diagnosis rates may increase with age. There is some evidence that the severity of PID might increase with the number of previous PID episodes. The proportion of women who become infertile because of tubal scarring from adhesions, and the proportion of pregnancies that are ectopic, appears to increases multiplicatively with number of previous PID episodes,³³ which is likely to be positively correlated with age, and it is likely that more severe PID has a higher chance of being diagnosed and hospitalised. This hypothesis is supported by the dramatic increase with age of hospital-diagnosed PID.

A second possibility is that the probability that a case of CT causes an episode of PID may be higher in older women, perhaps owing to more previous episodes of CT, other STIs and PID.¹⁰⁷

Finally, the proportion of PID that is caused by CT may decline with age (see Chapter 7), possibly because of differences in the age distributions of the other risk factors for PID. Facultative and anaerobic bacteria are frequently isolated from women with PID; although often present in the normal vaginal flora, they occur more often and in increased concentrations, in women with BV,⁸² which has been associated with PID. Interestingly, isolation of endogenous bacteria from the upper genital tract appears more common in older women and may be associated with severe suppurative disease, including tubo-ovarian abscess and recurrent PID.^82,108 Although BV does not appear to be associated with age, it is associated with increasing lifetime number of sexual partners.^94,109

These three different explanations would have quite different public health implications. Analyses in subsequent chapters throw some light on the issues, but we do not believe it can be resolved on the basis of existing evidence, and this is addressed in the research recommendations in Chapter 12.

Salpingitis

Much of our knowledge of the impact of PID on reproductive health is based on the Swedish Lund study,^33,34,84,110 which recruited PID cases admitted to hospital between 1960 and 1984, and followed them forward to observe reproductive outcomes over a median 8-year period. The study was based on laparoscopic examination. Women with macroscopic inflammation of the fallopian tubes, ‘salpingitis’, were distinguished from PID cases with no macroscopic inflammation at laparoscopy. The more severe the macroscopic salpingitis, the greater the risk of adverse reproductive outcomes. Remarkably, the incidence of TFI and EP in the control group, those admitted to hospital with PID but with no salpingitis on laparoscopy, appeared to be no different from the general population, even though a lower genital tract infection was identified in the majority of cases.³³ It is likely that many of these women would have had endometritis or microscopic inflammation of the fallopian tubes but some will have had other pathology or causes for their pelvic pain.^83,111,112 Incidence of EP and TFI among women with salpingitis depended on age, the number of clinical PID episodes – and thus presumably on the number of salpingitis episodes – and on the severity of the salpingitis on admission (see Chapters 9 and 10).

These results raise the question: what proportion of clinically diagnosed PID is accompanied by an underlying macroscopic salpingitis? Unfortunately for our purposes, laparoscopy is no longer a routine procedure in examination of PID, so it is not possible to match up the definitions of PID and salpingitis employed in the Lund study^33,34,84,110 to current practice or to recent research studies. However, we can deduce from the fact that they were hospitalised that the cases of PID recruited into the Lund study^33,34,84,110 would be at the more severe end of the spectrum. What is known is that the proportion of clinical PID cases with salpingitis fell during the course of the Lund study^33,34,84,110 from an initial 80% down towards 60% among those recruited at the end of the study.¹¹⁰ In the analyses we present in Chapters 10 and 11, we have assumed that the proportion of PID cases with salpingitis has continued to fall.

Finally, there is little quantitative information on whether the organism causing PID impacts on the probability or extent of reproductive damage. In the Lund study^33,34,84,110 it was observed that the severity of CT salpingitis at laparoscopy was generally greater than would be expected from the relatively benign clinical picture.¹⁰¹ For PID with salpingitis (including that caused by CT), delay of treatment for more than 3 days from onset of symptoms increases the likelihood of tubal damage.⁸⁴ Therefore, if the clinical symptoms of CT-related PID appear to be less severe than gonococcal PID, it is possible that women with chlamydia PID may present later than women with gonococcal PID,^{101,113–115} and as a result suffer more reproductive damage. The same comments apply to women with asymptomatic PID.

Ectopic pregnancy

In a normal pregnancy the fertilised egg implants in the uterus, where it divides and develops into an embryo and, eventually, a fetus. An ectopic pregnancy is one in which the egg implants outside the uterus, most often in the Fallopian tubes. EP is an important cause of maternal morbidity and, occasionally, mortality.¹¹⁶ It usually presents with acute symptoms including pelvic pain and vaginal bleeding, although increasingly EPs are diagnosed before the onset of symptoms, allowing early, conservative treatment. Prevention of maternal morbidity and mortality relies on early diagnosis and appropriate management, which is almost invariably in a specialist setting.¹¹⁶

Among the common causes of EP are fallopian tube damage because of salpingitis, surgery and smoking.¹¹⁶ In a normal pregnancy, cilia in the Fallopian tubes carry the fertilised egg towards the uterus. If the cilia are damaged, the egg may become lodged in the tube. PID is an inflammatory process that can result in the development of scar tissue in the tubes. If both tubes become entirely blocked then fertilisation cannot take place at all. This constitutes a single common pathway leading from salpingitis to both EP and to TFI (which will be exploited in the modelling in Chapters 9 and 10).^98,116

Data on overall EP rates in England are based on HES (HES code O00).¹¹⁷ If these are compared with conception rates (maternities plus abortions),¹¹⁸ it is evident that the EP rate, defined as the number of EPs divided by the number of conceptions, has remained remarkably constant between 2000 and 2009 (Figure 3), at around 1% of conceptions. Similar rates have been reported in France, although there was evidence that a fall in intrauterine device (IUD)-related EPs had masked a rise in EPs caused by salpingitis.¹¹⁹ EP rates reported in the USA have been somewhat lower, but, again, have remained stable over recent years.¹²⁰

FIGURE 3

Proportion of conceptions that result in EP, by year and age group.

Although the overall conception rate falls steeply from the age of 30 years onwards, the proportion of conceptions that are EPs rises with age (see Figure 3). The same phenomenon has been observed many times.^120–122 This points to the role of cumulative exposure to the causal factors, whether these are smoking or tubal damage, which can only increase with age.

The fact that EP risk links to cumulative exposure to risk factors, including salpingitis, rather than to, say, incident exposure, is critical to understanding the role of PID.¹²³ Egger et al.¹²¹ report an ecological analysis of EP rates and CT prevalence by age and location in Uppsala county, Sweden. They establish that the correlation between CT prevalence and EP rates is strongest in 20- to 24-year-olds, and that it becomes increasingly weak in the 25- to 29-year-old, 30- to 34-year-old, and finally the 35- to 39-year-old groups. At the same time the regression of EP rate against CT prevalence becomes increasingly steep, which would not be expected if variation in EP rates was driven by variation in CT incidence. However, these phenomena can be readily explained if cumulative salpingitis incidence is the variable determining EP rates. Most CT occurs in the younger age groups, but current incidence and prevalence become increasingly poor predictors of cumulative exposure to CT as age increases. On the other hand, the regression against recent incidence becomes increasingly steep with age – although with increasing error about the regression – because cumulative incidence is higher per unit of current prevalence. These ecological observations provide further support for the conclusions we draw from Figure 3 in the previous paragraph.

Another important feature of the relation between salpingitis and EP is the strong ‘dose–response’ relationship reported in prospective studies following women with salpingitis. The Lund studies,^33,110,124 in particular, followed women for an average of approximately 8 years from hospitalisation for PID. The proportion of women whose first subsequent pregnancy was an EP increased systematically with severity as determined by laparoscopy, and also with the number of previous salpingitis episodes.¹²⁴ Among the women who subsequently conceived, those with two, or three or more previous episodes of salpingitis had a non-linear increase in risk of EP (see Chapter 9). A similar pattern of findings was reported for TFI.³³ A record linkage study in the USA following high-risk women with a diagnosed CT infection over an average 6-year period again reported a similar increase in risk of EP, but this time in relation to the number of previous CT infections rather than the number of PID episodes.¹⁰⁷ These studies constitute some of the strongest evidence for a causal link between salpingitis (PID) and EP. Of particular significance is the fact that the dose–response relation is exactly what would be expected on the basis of cumulative exposure to risk factors. In addition, the similarity of the ‘dose–response’ effect attaching to CT and salpingitis suggests that the risk of EP may be approximately the same whatever the cause of the salpingitis, although the agreement could be coincidental if the aetiology changes with age in a way that approximately cancels out any differences.

Tubal factor infertility

Women are usually considered ‘infertile’ if they have tried to have a baby for ≥ 1 year and have been unsuccessful. Defined in this way, infertility is clearly not an absolute condition, as only 85% of couples succeed in conceiving within 1 year, whereas 95% may succeed within 3 or 4 years.¹²⁵ This immediately reveals a major difficulty in the study of infertility, as apparently minor changes in definition readily generate threefold changes in the number considered infertile.

Infertility is a complex construct: the infertile population includes both couples who are sterile, and couples who are subfertile. The former cannot conceive, but the latter can, if given enough time. Population surveys of infertility in the UK^126–128 distinguish between primary unresolved failure to conceive, between 2% and 3.5%, representing women who have not conceived at the end of their reproductive life, and secondary infertility, which includes women who have conceived at least once, but who subsequently experience difficulty in conceiving. Secondary infertility must be included in our study because it is quite possible for salpingitis to be acquired by women who have already had children.

Surveys reporting secondary infertility have reported the numbers who have experienced at least two years of being unable to conceive.^126–128 According to one study,¹²⁶ the secondary infertility group included women who had already had one or two, and in one case eight, previous children. The 2–3.5% estimates of secondary infertility from these surveys are therefore probably an overestimate, but the extent of the overestimate depends on the distribution of duration of infertility.

A further problem is the difficulty of estimating the proportion of women who are infertile at any particular age. Not only is it necessary to ensure that their infertility has lasted sufficiently long to meet a specified definition, but infertility can only manifest itself, and thus be diagnosed, in women who are trying to become pregnant, although the underlying condition may have been present for a long time. This contrasts with the study of EP where reliable age-specific incidence data are available from routine hospital statistics.

To avoid these problems in this monograph we have followed a strategy that has been adopted in a number of epidemiological studies of infertility in the UK,^126–129 which is to focus attention on the proportion of the population who are infertile, which specifically means unable to conceive, at the end of their reproductive life, which we take to be the age of 44 years (see Chapter 10).

Tubal factor infertility is one of several causes of female infertility (Figure 4).¹³⁰ According to a 2006 survey by the Human Fertilisation and Embryology Authority (HFEA), 22% of those being treated by in vitro fertilisation (IVF) have TFI (Table 3), but a further 30% of infertility in women undergoing treatment is unexplained.¹³¹

FIGURE 4

Types of infertility treated 2000–7. Source HFEA.

TABLE 3

Tubal factor infertility by age, 2002

The Lund studies¹²⁴ established the key relationship between PID and TFI, which parallels the relationship between EP and TFI. A higher rate of TFI was observed only in PID cases with confirmed salpingitis on laparoscopy, and the risk increased with the number of subsequent PID episodes. Critically, none of the 601 control group women with PID who were not confirmed as having salpingitis went on to have TFI. A difference between EP and TFI, however, is that TFI can occur only following an infective process,³⁵ namely salpingitis; EP on the other hand has a multifactorial aetiology.

There is an extensive literature on the role of CT in TFI. Much of it consists of retrospective studies demonstrating a higher prevalence of serum antibody to CT in TFI cases compared with non-TFI controls. Because of the poor sensitivity and specificity of the assays, the imprecise characterisation of their diagnostic accuracy, and the variation in cut-offs, these studies have not generated reliable quantitative estimates of the role of CT in TFI. However, a number of these retrospective studies have reported marked differences in the titre distributions in antibody-positive TFI cases and controls (see Chapter 11). The higher prevalence of high titres in TFI cases constitutes powerful evidence of the causal role of CT.

Chronic pelvic pain

Chronic pelvic pain is a well-recognised complication of PID, which has a significant morbidity.^33,132 It is defined as non-menstrual pain of ≥ 6 months’ duration that is severe enough to cause functional disability or require medical or surgical treatment. Chronic pelvic pain is common in women, with an estimated prevalence of 3.8% in women aged 15–73 years in the USA. Often the aetiology is not clear. There are many physical disorders of the reproductive tract, gastrointestinal system, urological, musculoskeletal system and psychoneurological system that may be associated with chronic pelvic pain in women,¹³² and these may often co-exist. Although adhesions resulting from pelvic inflammatory disease are diagnosed in about 25% of women undergoing investigation, it is not clear whether these are always the cause of the pain.¹³² The prevalence of adhesions in women with chronic pelvic pain will vary depending on the population studied. Most information is obtained from women attending secondary and tertiary care, who are likely to experience more severe forms and may not be representative of women with chronic pelvic pain in the community. An accurate diagnosis of pelvic adhesions is only possible following laparoscopy and, even then, it is not possible to ascertain the cause, as the infectious agent will have usually resolved prior to investigation.

We have therefore chosen not to investigate chronic pelvic pain because of the paucity of data on the role of PID, and the lack of evidence on the contribution of CT.

Premature delivery, neonatal pneumonia and conjunctivitis

There is conflicting evidence that CT may result in miscarriage and/or premature labour. One hypothesis is that adverse pregnancy outcomes may only occur following primary infection. No studies have investigated this since 1990. In view of the limited and conflicting data on adverse pregnancy outcomes, we decided not to include this in our work plan.

Chlamydia trachomatis is transmitted to approximately half of children of mothers infected with CT and about 10–50% of these develop neonatal conjunctivitis and/or pneumonia. This was recently reviewed by Thorne¹³³ for the NSC. Data in the UK are based on those presenting to hospital and thus biased towards severe and prolonged cases. Neonatal infection with chlamydia is usually asymptomatic, and the most common manifestations (conjunctivitis and pneumonia) are non-specific. Furthermore, chlamydial disease is usually of a mild to moderate nature and easily treated or is often self-limiting. For these reasons, perinatal and neonatal outcomes have not been included in this study.