Menstrual Periods Began Again After Having a Leep Procedure
Background. A delayed time to pregnancy was recently reported for women who had a loop electrosurgical excision procedure (LEEP) to remove cervical intraepithelial neoplasia (CIN) grade two or 3. The objective of the current report was to determine if treatment of CIN with LEEP is associated with decreased levels of anti-Müllerian hormone (AMH), a marker of ovarian reserve. Methods. AMH levels were measured in 18 women treated with LEEP and xviii age-matched controls, who had colposcopy only and did not require LEEP. Cases and controls had their blood drawn at report entry time zero and again six months later. Results. The mean AMH level decreased significantly from baseline to follow-up; however, no significant differences were observed when stratifying by LEEP status, suggesting that both groups experienced a similar decrease in AMH levels during the follow-upward catamenia. Although women treated with LEEP had lower overall AMH levels than controls at both baseline and follow-upward, these differences were non statistically significant. Determination. Overall, the delayed time to pregnancy observed in women treated with LEEP is probable not due to a LEEP-associated subtract in ovarian reserve as measured past AMH; thus, other mechanism are responsible for the delayed time to pregnancy associated with LEEP.
ane. Introduction
In the United States (US), pap smears to detect precancerous cervical intraepithelial neoplasia (CIN) are an integral part of a woman's health care regimen. Every bit a result of screening, women with biopsy confirmed CIN2 or CIN3 routinely undergo loop electrosurgical excision procedure (LEEP) to remove cervical dysplasia, equating to approximately half a one thousand thousand LEEP procedures in the United states each twelvemonth (American Cancer Society/[i]). Considering nigh women requiring LEEP are of child begetting historic period, preserving fertility is paramount. However, the impact of LEEP on fertility is not established. Although a few small studies from the early 1990s ended that LEEP had no effect on futurity fertility, these studies were non designed to directly address this question and lacked information on potentially misreckoning factors such as a history of infertility [2–4].
In a recent written report of time to pregnancy following LEEP and other cervical surgical procedures, Spracklen et al. reported that women with a history of LEEP were significantly more probable to require more than than 12 months to conceive a pregnancy resulting in a live birth (odds ratio two.47, 95% Conviction Interval i.ten–five.55) when compared to similar women with no history of cervical surgery [5]. While this finding suggests that LEEP is associated with reduced fertility, the underlying mechanisms responsible for this link take not yet been identified. Because LEEP is a common process, investigating underlying biological causes of a delayed time to conception of a viable pregnancy for women with a history of LEEP is of interest.
Anti-Müllerian hormone (AMH) is an established mark of ovarian reserve, which is tightly linked to female fertility. Therefore, measuring AMH levels before and afterwards LEEP could determine if the procedure affects this vital component of female fertility. AMH is a modest peptide hormone inside the TGF-beta family that is currently used to diagnose subfertility/infertility, master ovarian insufficiency (POI), and polycystic ovarian syndrome (PCOS), amidst other disorders. In addition, AMH is now the leading predictor of in vitro fertilization (IVF) success [3, 4, half-dozen–8]. Recent literature has demonstrated that AMH is a more reliable measure out of fertility than follicle-stimulating hormone (FSH) [4, 7–11]. Normal ranges of AMH are broad and the normal range divers specifically for the AMH assay used in this study is 1–8 ng/mL [12]. For healthy women of reproductive historic period, an AMH level less than 1 ng/mL signifies impaired fertility due to an inadequate ovarian reserve [12]. AMH can be measured at whatever point in the menstrual bicycle as information technology does not appear to fluctuate significantly throughout the cycle, unlike FSH, which must exist measured on mean solar day three of the menstrual cycle [vi–8]. Because the merely cells that produce AMH in women are the granulosa cells in the ovaries, AMH is non subject area to the feedback mechanisms of the hypothalamic-pituitary-gonadal axis [6, 8]. In women, AMH levels rising just prior to puberty, remain elevated throughout a woman's top reproductive years, then significantly decline to undetectable levels in the years prior to menopause, indicative of a loss of fertility [13, 14].
Elevated levels of cytokines resulting from endometriosis, sexually transmitted infections, pelvic inflammatory affliction, and other gynecologic disorders take been reported to negatively influence fertility. We hypothesize that the nonspecific inflammation and associated cytokine production resulting from LEEP may crusade indirect damage to the urogenital microenvironment, thereby impairing ovarian reserve [12, 15]. Inflammatory cytokines are capable of negatively affecting ovulation, hormones required for reproduction, sperm and egg quality, and implantation [12, xv, xvi]. To investigate the potential causes for the described decrease in fertility later on LEEP [5], nosotros designed a report to compare levels of the fertility marking, AMH, in women of reproductive age who had LEEP (CIN2/3) and a like group of women who did not require LEEP (<CIN2) following colposcopy.
2. Materials and Methods
two.one. Subjects
The subjects for this assay participated in a longitudinal report to assess the affect of LEEP on the immunologic properties of cervical fungus. Of these subjects, merely a subset was used for our current study assessing the effect of LEEP on AMH levels (Supplemental Effigy 1 in Supplementary materials bachelor online at http://dx.doi.org/10.1155/2014/875438). This study recruited women of reproductive historic period who had underwent colposcopy at the Academy of Iowa from 2009-2010. Written informed consent was obtained to let collection and analysis of demographic information, clinical data, cervical secretions and blood samples. All protocols and informed consent procedures were approved past the University of Iowa Institutional Review Board. Women eligible to participate were xviii–38 years old, were not significant, had no prior history of cervical surgery, had regular menstrual cycles of 21–35 days, and had no history of D&C, induced abortion, cervical dysplasia, cancer, HIV/AIDS, or autoimmune disease (e.g., rheumatoid arthritis, lupus, and multiple sclerosis). Samples were non collected from women who used oral steroids inside the past 2 weeks or inhaled steroids within the by 24 hours; used emergency contraception in the past 30 days; douched within the past 48 hours; engaged in vaginal intercourse inside the past 48 hours; currently had a vaginal or sexually transmitted infection other than HPV; became pregnant during the written report; or were currently menstruating.
A total of 63 qualifying subjects who required a LEEP procedure (cases: CIN2/3) and 49 subjects who did not require cervical surgery (controls: <CIN2) enrolled in the study. A subset of the enrolled women (19 LEEP and 28 No LEEP controls) besides consented to provide blood samples at baseline and at follow-up for future analysis. Cases and controls had their claret drawn at report entry time cipher (baseline), which was merely prior to the L EEP procedure for cases, and again 6 months afterwards (follow-upwardly). Blood was collected during the same phase of the menstrual bike for each private woman at both time points.
For this report, we selected all 19 available cases, defined as women who were treated with LEEP afterward colposcopy and 19 historic period-matched controls, who did not receive LEEP after colposcopy. Cases and controls were historic period-matched inside two years because AMH levels are known to decrease with age following meridian fertility [xiii, 17–19]. Five of 19 cases and 2 of 19 controls were smokers at report entry and through the follow-up period. Additionally, eighteen/19 cases and 15/19 controls were using hormonal contraception at written report entry and continued to practise so throughout the duration of the study. One case-control pair was eliminated because of an approximately 300% increase in AMH levels from baseline to follow-upwardly measurements. All other subjects had less than a 60% modify in AMH levels from baseline to follow-up. Reported analyses were performed on the remaining eighteen cases and 18 age-matched controls.
ii.2. AMH ELISA Assay
AMH was measured in the serum of all subjects using the sensitive ( ng/mL) Gen Ii AMH ELISA from Beckman Coulter, Inc. (United states) at Frederick National Laboratory for Cancer Research (FNLCR) (Frederick, Dr.) according to the manufacture'southward protocol. This analysis is used clinically in Europe and in the U.s.a.. Excellent intra- and interplate reproducibility has been reported in the literature for this analysis [20]. The assay reproducibility was confirmed in the FNLCR lab where the assay consistently performed well with intra- and interplate variability of <x%. For this study, case and control samples were randomized and run in duplicate in three divide AMH assays.
2.3. Statistical Analysis
Univariate, bivariate, and stratified analyses were conducted to appraise the change in AMH levels inside the LEEP and No LEEP groups over the 6-month follow-upward period. Paired -tests were used to compare mean AMH levels from baseline to follow-up. Analysis of variance was performed to appraise for differences in the mean change in AMH levels between case and control groups. Percent modify in AMH levels between baseline and follow-up was calculated for all subjects. Although the estimated percent change in AMH in a good for you population of reproductive age over a half dozen-month period is 7.5% [21], the variability of the assay tin range to almost x%. For this reason, we defined alter in AMH levels over the follow-upward period equally being equivalent to no pregnant percent alter in AMH. All information analyses were conducted using SAS software, version 9.3 for Microsoft, SAS Institute Inc., Cary, NC, United states. Graphs were generated using GraphPad Prism 4.
3. Results and Discussion
iii.one. Results
The mean AMH levels for all subjects by LEEP status are shown in Table one. Mean levels of AMH declined significantly from baseline to the 6-month follow-upward amidst all 36 subjects (3.56 versus three.07 ng/mL, ). When women were stratified based on LEEP status, both groups had a like subtract in AMH, although the decrease was not statistically meaning at the level (LEEP, ; No LEEP, ). Women from the LEEP group had lower levels of AMH compared to the No LEEP grouping at both baseline (LEEP: 3.02 ng/mL versus No LEEP: 4.09 ng/mL) and follow-up (LEEP: 2.66 ng/mL versus No LEEP: 3.48 ng/mL), though these differences were not statistically significant. At the half-dozen-month follow-up visit, two women in each group had AMH levels indicative of an impaired ovarian reserve (<1 ng/mL) (data not shown). Figure one shows the AMH measurements for each subject at baseline and follow-up stratified by LEEP condition.
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| Abbreviations: AMH: anti-Müllerian hormone; LEEP: loop electrosurgical procedure. aPaired -examination for baseline versus follow-upwards AMH levels (deviation between the means). bAnalysis of variance to test for difference between mean at baseline or at follow-up for LEEP versus No LEEP. cBaseline mean AMH value minus follow-upward mean AMH value. Med.: median; Min.: minimum; Max.: maximum. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Figure two(a) displays the baseline and follow-up AMH levels for each subject. Overall, AMH levels decreased over the six-month follow-up period for nearly women. Although a reduction in AMH over time is expected, several subjects' levels decreased in backlog of the 9.9% expected based on historic period and assay variability [21]. On average, AMH levels in LEEP and No LEEP subjects decreased similarly (−10.47% versus −nine.38%, resp.) (Effigy two(b)). There was not a pregnant difference in the overall decrease in AMH levels in women who had tissue removed by LEEP versus their age-matched controls who had no tissue removed; however, more subjects from the LEEP group had a notable decrease in AMH levels (>9.9%) compared to No LEEP subjects (12 versus 9) (Table 2). Similar numbers of LEEP and No LEEP subjects had no significant modify in AMH levels (<±9.9%) (3 versus 4), though virtually twice as many control subjects had an increase in AMH levels (>9.9%) when compared to case subjects (5 versus 3).
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| Abbreviations: AMH: anti-Müllerian hormone; LEEP: loop electrosurgical excision procedure. aNo change is equivalent to an AMH change of ±9.9%. | |||||||||||||||||||||||||||
(a)
(b)
iii.ii. Discussion
In the first study to investigate underlying mechanisms of reduced fertility afterward LEEP, our findings advise that AMH is not affected by LEEP and, therefore, another mechanism is responsible for the delayed time to pregnancy observed in women treated with LEEP. Furthermore, innate and environmental factors can cause the levels of AMH to vary broadly from woman to woman, fifty-fifty of the same age, so the fairly big range of AMH levels observed in our written report is not surprising [17]. Over the vi-month follow-upwardly period, the percent decrease in AMH levels in women who had LEEP versus those observed in age-matched controls was quite comparable and non statistically significant. These data propose that LEEP does not significantly affect ovarian reserve.
An interesting observation from this small study is that the mean and median AMH levels for LEEP subjects, all of whom had been diagnosed with CIN2/3 cervical lesions, tended to be lower than those among control women who had less than CIN2 pathology and did not require LEEP. This is in agreement with our finding that women whose AMH levels increased over the 6-month follow-up period were less likely to be in the LEEP group (CIN2/three). There may exist an increased take chances of cervical disease for women with lower AMH levels since mounting show demonstrates the anticancer effects of AMH in vitro and in vivo [13]. Information technology is possible that AMH may play a part in cancer control in humans [13]. In recent years, AMH has been investigated equally an anti-cancer agent, in add-on to its better known role as a predictor of female fertility.
Because pathology reports and HPV Deoxyribonucleic acid information were unavailable, further studies are needed to investigate the possibility of a directly association between HPV-associated cervical disease and AMH. Small sample size is some other limitation of the electric current study. To address these limitations nosotros are conducting a larger study to more directly investigate if lower AMH levels tin serve as a hazard factor for cervical illness.
Factors known to negatively affect AMH levels include smoking, chemotherapy, radiations, and any surgery removing or disturbing the ovaries [22–25]. It is largely reported that hormonal contraceptives do not have a significant upshot on AMH levels and the levels can exist measured at any time during the menstrual cycle; all the same, these problems are still debated in the literature.
Our study was controlled for whatever potential AMH flux every bit a issue of cycling as AMH was measured during the same phase of the menstrual wheel for each subject's baseline and follow-up visits. Withal, AMH was not measured during the aforementioned phase of the menstrual cycle for all case and control subjects on the whole. Factors that may atomic number 82 to an increase in AMH product include PCOS, granulosa cell ovarian tumors, smoking cessation, and increased sun exposure/Vitamin D levels [26–30]. It is likely that there are likewise unidentified intrinsic and environmental factors that affect AMH levels. These unknown factors deserve farther investigation equally they could be determinants for the wide ranges of AMH in women of similar age and may contribute to unexpectedly loftier AMH variability across the 6-calendar month period for some subjects.
Despite the modest sample size and data of yet unknown factors that affect AMH levels, this study has several strengths, namely, the careful screening process designed to exclude women with misreckoning factors including history of infertility, vaginal infections (other than HPV), and irregular menstrual cycles, which could bias example and control populations.
iv. Conclusions
In summary, this written report suggests that the recently reported increased time to pregnancy among women who have had cervical surgery is non the issue of a LEEP-induced subtract in AMH, though the reason why in that location is delayed fertility in women treated with LEEP remains unknown [five]. Direct damage to the ovaries and ovarian reserve resulting from LEEP is doubtful. LEEP-induced endometriosis may inhibit joining of sperm and egg as would the destruction of mucus-producing cervical glands, which aid in sperm capitulation and subsequent fertilization [16, 31]. Additionally, direct or indirect physical damage to cervical tissue during LEEP could cause cervical stenosis, impair embryonic implantation, or consequence in an unfavorable microenvironment for pregnancy [15, 16, 32, 33]. Because half a 1000000 women of reproductive age are treated with LEEP each twelvemonth in the United States alone, future research is needed to investigate the underlying causes for delayed fourth dimension to pregnancy with LEEP [5].
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
Acknowledgments
The authors thank Mary Cherrico for contributions to the data drove. This project has been funded in whole or in part with federal funds from the National Cancer Plant, National Institutes of Health, under Contract no. HHSN261200800001E. The content of this paper does non necessarily reverberate the views or policies of the Section of Health and Human being Services, nor does the mention of trade names, commercial products, or organizations imply endorsement past the The states Government. This project was too funded past the NIH Grant 5R21A1068111-02 to Audrey F. Saftlas at the Academy of Iowa, Iowa City, IA.
Supplementary Materials
Supplemental Figure i. Flow chart of the LEEP study subject recruitment process. A total of 112 participants completed the study. There were 63 qualifying subjects who required a LEEP process (cases: CIN2/3) and 49 subjects who did not crave cervical surgery (controls: CIN2). A subset of the enrolled women (19 LEEP and 28 No LEEP controls) also consented to provide blood samples at baseline and at follow-up visits for hereafter analysis. For the current study we selected all 19 available cases and 19 historic period-matched controls. One case-control pair was eliminated because of an approximately 300% increase in AMH levels from baseline to follow-up measurements.
- Supplementary Figure
Copyright
Copyright © 2014 Martha Thousand. Sklavos et al. This is an open admission article distributed under the Creative Eatables Attribution License, which permits unrestricted apply, distribution, and reproduction in whatsoever medium, provided the original work is properly cited.
Source: https://www.hindawi.com/journals/bmri/2014/875438/
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